Branched oligonucleotides

ABSTRACT

Provided herein are branched oligonucleotides exhibiting efficient and specific tissue distribution, cellular uptake, minimum immune response and off-target effects, without formulation.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/419,593, filed Jan. 30, 2017, which claims priority to U.S.Provisional Patent Application Nos. 62/289,268, filed Jan. 31, 2016, and62/317,113, filed Apr. 1, 2016. The contents of the aforementionedapplications are incorporated by reference herein for all purposes.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. GM108803awarded by the National Institutes of Health. The Government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 17, 2019, isnamed 612182 UM9-213DIV ST25.txt and is 10,310 bytes in size.

TECHNICAL FIELD

This disclosure relates to novel branched oligonucleotides designed toachieve unexpectedly high efficacy, uptake and tissue distribution.

BACKGROUND

Therapeutic oligonucleotides are simple and effective tools for avariety of applications, including the inhibition of gene function. Anexample of such inhibition is RNA interference (RNAi). The promise ofRNAi as a general therapeutic strategy, however, depends on the abilityto deliver small RNAs to a wide range of tissues. Currently, smalltherapeutic RNAs can only be delivered effectively to liver. Thereremains a need for self-delivering siRNA, and therapeuticoligonucleotides in general, that exhibit minimal immune response andoff-target effects, efficient cellular uptake without formulation, andefficient and specific tissue distribution.

SUMMARY

Accordingly, the present disclosure provides branched oligonucleotides(“compounds of the invention”) exhibiting unexpected improvement indistribution, in vivo efficacy and safety.

In a first aspect, provided herein is a branched oligonucleotidecompound comprising two or more nucleic acids, the nucleic acids areconnected to one another by one or more moieties selected from a linker,a spacer and a branching point.

In an embodiment, the branched oligonucleotide comprises 2, 3, 4, 6 or 8nucleic acids.

In an embodiment of the branched oligonucleotide, each nucleic acid issingle-stranded and has a 5′ end and a 3′ end, and each nucleic acid isindependently connected to a linker, a spacer, or a branching point atthe 5′ end or at the 3′ end.

In an embodiment, each single-stranded nucleic acid independentlycomprises at least 15 contiguous nucleotides. In an embodiment, theantisense strand comprises at least 15, at least 16, at least 17, atleast 18, at least 19, or at least 20 contiguous nucleotides, and hascomplementarity to a target.

In an embodiment, each nucleic acid comprises one or morechemically-modified nucleotides. In an embodiment, each nucleic acidconsists of chemically-modified nucleotides.

In an embodiment of the branched oligonucleotide, each nucleic acid isdouble-stranded and comprises a sense strand and an antisense strand,the sense strand and the antisense strand each have a 5′ end and a 3′end. In an embodiment, each double-stranded nucleic acid isindependently connected to a linker, spacer or branching point at the 3′end or at the 5′ end of the sense strand or the antisense strand.

In an embodiment, the sense strand and the antisense strand eachcomprise one or more chemically-modified nucleotides. In an embodiment,the sense strand and the antisense strand each consist ofchemically-modified nucleotides. In an embodiment, the sense strand andthe antisense strand both comprise alternating 2′-methoxy-nucleotidesand 2′-fluoro-nucleotides. In an embodiment, the nucleotides atpositions 1 and 2 from the 5′ end of the sense and antisense strands areconnected to adjacent nucleotides via phosphorothioate linkages. In anembodiment, the nucleotides at positions 1-6 from the 3′ end, orpositions 1-7 from the 3′ end, are connected to adjacent nucleotides viaphosphorothioate linkages.

In an embodiment, the branched oligonucleotide further comprises ahydrophobic moiety. In a particular embodiment, the hydrophobic moietyis attached to one or more terminal 5′ positions of the branchedoligonucleotide compound. The hydrophobic moiety may be comprised withinone or more 5′ phosphate moieties. In certain embodiments, thehydrophobic moiety comprises an alkyl or alkenyl moiety (e.g., an alkylor alkenyl chain, or a saturated or unsaturated fatty acid residue), avitamin or cholesterol derivative, an aromatic moiety (e.g., phenyl ornaphthyl), a lipophilic amino acid or a combination thereof. Certainembodiments of hydrophobic moieties, and strategies for synthesizinghydrophobically modified branched oligonucleotide compounds, aredepicted in FIG. 44.

In an embodiment of the branched oligonucleotide, each linker isindependently selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof any carbon oroxygen atom of the linker is optionally replaced with a nitrogen atom,bears a hydroxyl substituent, or bears an oxo substituent.

In a second aspect, provided herein is a compound of formula (I):

L—(N)_(n)  (I)

L is selected from an ethylene glycol chain, an alkyl chain, a peptide,RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, anamide, a triazole, and combinations thereof, formula (I) optionallyfurther comprises one or more branch point B, and one or more spacer S;B is independently for each occurrence a polyvalent organic species orderivative thereof; S is independently for each occurrence selected froman ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, aphosphate, a phosphonate, a phosphoramidate, an ester, an amide, atriazole, and combinations thereof; N is an RNA duplex comprising asense strand and an antisense strand, the sense strand and antisensestrand each independently comprise one or more chemical modifications;and n is 2, 3, 4, 5, 6, 7 or 8.

In an embodiment, the compound of formula (I) has a structure selectedfrom formulas (I-1)-(I-9) of Table 1.

TABLE 1 N—L—N (I-1) N—S—L—S—N (I-2)

(I-3)

(I-4)

(I-5)

(I-6)

(I-7)

(I-8)

(I-9)

In an embodiment, each antisense strand independently comprises a 5′terminal group R selected from the groups of Table 2.

TABLE 2

R¹

R²

R³

R⁴

R⁵

R⁶

R⁷

R⁸

In an embodiment, the compound of formula (I) the structure of formula(II):

X, for each occurrence, independently, is selected from adenosine,guanosine, uridine, cytidine, and chemically-modified derivativesthereof; Y, for each occurrence, independently, is selected fromadenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; — represents a phosphodiester internucleosidelinkage; ═ represents a phosphorothioate internucleoside linkage; and ≡represents, individually for each occurrence, a base-pairing interactionor a mismatch.

In an embodiment, the compound of formula (I) has the structure offormula (III):

X, for each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; X, for each occurrence, independently,is a nucleotide comprising a 2′-O-methyl modification; Y, for eachoccurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; and Y, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification.

In an embodiment, the compound of formula (I) has the structure offormula (IV) (SEQ ID NOS 1 and 2, respectively, in order of appearance):

A is an adenosine comprising a 2′-deoxy-2′-fluoro modification; A is anadenosine comprising a 2′-O-methyl modification; G is an guanosinecomprising a 2′-deoxy-2′-fluoro modification; G is an guanosinecomprising a 2′-O-methyl modification; U is an uridine comprising a2′-deoxy-2′-fluoro modification; U is an uridine comprising a2′-O-methyl modification; C is an cytidine comprising a2′-deoxy-2′-fluoro modification; and C is an cytidine comprising a2′-O-methyl modification.

In an embodiment, the compound of formula (I) has the structure offormula (V):

X, for each occurrence, independently, is selected from adenosine,guanosine, uridine, cytidine, and chemically-modified derivativesthereof; Y, for each occurrence, independently, is selected fromadenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; — represents a phosphodiester internucleosidelinkage; ═ represents a phosphorothioate internucleoside linkage; and ≡represents, individually for each occurrence, a base-pairing interactionor a mismatch.

In an embodiment, the compound of formula (I) has the structure offormula (VI):

X, for each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; X, for each occurrence, independently,is a nucleotide comprising a 2′-O-methyl modification; Y, for eachoccurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; and Y, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification.

In an embodiment, the compound of formula (I) has the structure offormula (VII) (SEQ ID NOS 3 and 4, respectively, in order ofappearance):

A is an adenosine comprising a 2′-deoxy-2′-fluoro modification; A is anadenosine comprising a 2′-O-methyl modification; G is an guanosinecomprising a 2′-deoxy-2′-fluoro modification; G is an guanosinecomprising a 2′-O-methyl modification; U is an uridine comprising a2′-deoxy-2′-fluoro modification; U is an uridine comprising a2′-O-methyl modification; C is an cytidine comprising a2′-deoxy-2′-fluoro modification; C is an cytidine comprising a2′-O-methyl modification; Y, for each occurrence, independently, is anucleotide comprising a 2′-deoxy-2′-fluoro modification; and Y, for eachoccurrence, independently, is a nucleotide comprising a 2′-O-methylmodification.

In an embodiment of the compound of formula (I), L has the structure ofL1:

In an embodiment of L1, R is R³ and n is 2.

In an embodiment of the compound of formula (I), L has the structure ofL2:

In an embodiment of L2, R is R³ and n is 2.

In a third aspect, provided herein is a delivery system for therapeuticnucleic acids having the structure of formula (VIII):

L—(cNA)_(n)  (VIII)

L is selected from an ethylene glycol chain, an alkyl chain, a peptide,RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, anamide, a triazole, and combinations thereof, formula (VIII) optionallyfurther comprises one or more branch point B, and one or more spacer S;B is independently for each occurrence a polyvalent organic species orderivative thereof; S is independently for each occurrence selected froman ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, aphosphate, a phosphonate, a phosphoramidate, an ester, an amide, atriazole, and combinations thereof; each cNA, independently, is acarrier nucleic acid comprising one or more chemical modifications; andn is 2, 3, 4, 5, 6, 7 or 8.

In an embodiment, the compound of formula (VIII) has a structureselected from formulas (VIII-1)-(VIII-9) of Table 3:

TABLE 3 ANc—L—cNA (VIII-1) ANc—S—L—S—cNA (VIII-2)

(VIII-3)

(VIII-4)

(VIII-5)

(VIII-6)

(VIII-7)

(VIII-8)

(VIII-9)

In an embodiment, the compound of formulas (VIII) (including, e.g.,formulas (VIII-1)-(VIII-9), each cNA independently comprises at least 15contiguous nucleotides. In an embodiment, each cNA independentlyconsists of chemically-modified nucleotides.

In an embodiment, the delivery system further comprises n therapeuticnucleic acids (NA), each NA is hybridized to at least one cNA.

In an embodiment, each NA independently comprises at least 16 contiguousnucleotides. In an embodiment, each NA independently comprises 16-20contiguous nucleotides. In an embodiment, each NA comprises an unpairedoverhang of at least 2 nucleotides. In an embodiment, the nucleotides ofthe overhang are connected via phosphorothioate linkages.

In an embodiment, each NA, independently, is selected from the groupconsisting of: DNA, siRNAs, antagomiRs, miRNAs, gapmers, mixmers, orguide RNAs. In an embodiment, each NA is the same. In an embodiment,each NA is not the same.

In an embodiment, the delivery system further comprising n therapeuticnucleic acids (NA) has a structure selected from formulas (I), (II),(III), (IV), (V), (VI), (VII), and embodiments thereof described herein.

In an embodiment of the delivery system, the target of delivery isselected from the group consisting of: brain, liver, skin, kidney,spleen, pancreas, colon, fat, lung, muscle, and thymus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of Di-hsiRNAs. Black—2′-O-methyl,grey—2′-fluoro, red dash—phosphorothioate bond, linker—tetraethyleneglycol. Di-hsiRNAs are two asymmetric siRNAs attached through the linkerat the 3′ ends of the sense strand. Hybridization to the longerantisense strand creates protruding single stranded fullyphosphorthioated regions, essential for tissue distribution, cellularuptake and efficacy. The structures presented utilize teg linger of fourmonomers. The chemical identity of the linker can be modified withoutthe impact on efficacy. It can be adjusted by length, chemicalcomposition (fully carbon), saturation or the addition of chemicaltargeting ligands.

FIG. 2 shows a chemical synthesis, purification and QC of Di-branchedsiRNAs.

FIG. 3 shows HPLC and QC of compounds produced by the method depicted inFIG. 2. Three major products were identified by mass spectrometry assense strand with TEG (tetraethylene glycol) linker, di-branched oligoand Vit-D (calciferol) conjugate. All products where purified by HPLCand tested in vivo independently. Only Di branched oligo ischaracterized by unprecedented tissue distribution and efficacy,indicating that branching structure is essential for tissue retentionand distribution.

FIG. 4 shows mass spectrometry confirming the mass of the Di-branchedoligonucleotide. The observed mass of 11683 corresponds to two sensestrands attached through the TEG linker by the 3′ ends.

FIG. 5 shows a synthesis of a branched oligonucleotide using alternativechemical routes.

FIG. 6 shows exemplary amidite linkers, spacers and branching moieties.

FIG. 7 shows oligonucleotide branching motifs. The double-helicesrepresented oligonucleotides. The combination of different linkers,spacer and branching points allows generation of a wide diversity ofbranched hsiRNA structures.

FIG. 8 shows structurally diverse branched oligonucleotides.

FIG. 9 shows an asymmetric compound of the invention having foursingle-stranded phosphorothioate regions.

FIGS. 10A-10C show in vitro efficacy data. FIG. 10A—HeLa cells weretransfected (using RNAiMax) with Di-branched oligo at concentrationsshown for 72 hours. FIG. 10B—Primary cortical mouse neurons were treatedwith Di-branched oligo at concentrations shown for 1 week. mRNA wasmeasured using Affymetrix Quantigene 2.0. Data was normalized tohousekeeping gene (PPIB) and graphed as % of untreated control. FIG.10C—HeLa cells were treated passively (no formulation) with Di-siRNAoligo at concentrations shown for 1 week.

FIGS. 11A-11B show brain distribution of Di-siRNA or TEG only after 48hours following intra-striatal injection. Intrastriatal injection of 2nmols of (FIG. 11A) Di-branched oligo (4 nmols of correspondingantisense strand) or (FIG. 11B) TEG-oligo only. N=2 mice per conjugate.Brains collected 48 hours later and stained with Dapi (nuclei, blue).Red—oligo. The left side of brain in FIG. 11A appears bright red,whereas the left side of the brain in FIG. 11B only faintly red.

FIG. 12 shows that a single injection of Di-siRNA was detected bothipsilateral and contralateral to the injection site.

FIGS. 13A-13B show Di-hsiRNA wide distribution and efficacy in mousebrain. FIG. 13A—Robust Htt mRNA silencing in both Cortex and Striatum 7days after single IS injection (25 ug), QuantiGene®. FIG. 13B—Levels ofhsiRNA accumulation in tissues 7 days after injection (PNA assay).

FIGS. 14A-14C show wide distribution and efficacy throughout the spinalcord following bolus intrathecal injection of Di-hsiRNA. Intrathecalinjection in lumbar of 3 nmols Di-branched Oligo (6 nmols ofcorresponding antisense HTT strand). FIG. 14A—Robust Htt mRNA silencingin all region of spinal cord, 7 days, n-6. Animals sacrificed 7 dayspost-injection. Tissue punches taken from cervical, thoracic and lumbarregions of spinal cord. mRNA was quantified using Affymetrix Quantigene2.0 as per Coles et al. 2015. Data normalized to housekeeping gene,HPRT, and graft as percent of aCSF control. aCSF—artificial CSF. FIG.14B—Animals were injected lumbar IT with 75 ug of Cy3-Chol-hsiRNA,Cy-Di-hsiRNA. Chol-hsiRNAs shows steep gradient of diffusion fromoutside to inside of spinal cord. Di-hsiRNAs shows wide distributionthroughout the cord (all regions). Leica 10× (20 mm bar). Image ofDi-branched oligo in cervical region of spinal cord 48 hours afterintrathecal injection. Red =oligo, Blue =Dapi. FIG. 14C—Image ofDi-branched oligo in liver 48 hours after intrathecal injection. Red=oligo, Blue =Dapi.

FIGS. 15A-15C show branched oligonucleotides of the invention, FIG.15A—formed by annealing three oligonucleotides. The longer linkingoligonucleotides may comprise a cleavable region in the form ofunmodified RNA, DNA or UNA; FIG. 15B—asymmetrical branchedoligonucleotides with 3′ and 5′ linkages to the linkers or spacesdescribed previously. This can be applied the 3′ and 5′ ends of thesense strand or the antisense strands or a combination thereof; FIG.15C—branched oligonucleotides made up of three separate strands. Thelong dual sense strand can be synthesized with 3′ phosphoramidites and5′ phosphoramidites to allow for 3′-3′ adjacent or 5′-5′ adjacent ends.

FIG. 16 shows branched oligonucleotides of the invention with conjugatedbioactive moieties.

FIG. 17 shows the relationship between phosphorothioate content andstereoselectivity.

FIG. 18 depicts exemplary hydrophobic moieties.

FIG. 19 depicts exemplary internucleotide linkages.

FIG. 20 depicts exemplary internucleotide backbone linkages.

FIG. 21 depicts exemplary sugar modifications.

FIGS. 22A-22C depict Di-FM-hsiRNA. FIG. 22A—Chemical composition of thefour sub-products created from VitD-FM-hsiRNA synthesis and crudereverse phase analytical HPLC of the original chemical synthesis. FIG.22B—Efficacy of sub-products in HeLa cells after lipid mediated deliveryof hsiRNA. Cells were treated for 72 hours. mRNA was measured usingQuantiGene 2.0 kit (Affymetrix). Data are normalized to housekeepinggene HPRT and presented as a percent of untreated control. FIG. 22C—Asingle, unilateral intrastriatal injection (25 μg) of each hsiRNAsub-product. Images taken 48 hours after injection.

FIGS. 23A-23B show that Di-HTT-Cy3does not effectively induce silencingin the liver or kidneys following intrastriatal injection. FIG. 23Adepicts a scatter dot plot showing Htt mRNA expression in the liver oneweek post intrastriatal injection of Di-HTT-Cy3 compared to a negativecontrol (aCSF). FIG. 23B depicts a scatter dot plot showing Htt mRNAexpression in the kidney one week post intrastriatal injection ofDi-HTT-Cy3 compared to a negative control (aCSF).

FIGS. 24A-24B show that Di-HTT effectively silences HTT gene expressionin both the striatum and the cortex following intrastriatal injectionand that Di-HTT-Cy3 is slightly more efficacious than Di-HTT(unlabeled). FIG. 24A depicts a scatter dot plot showing Htt mRNAexpression in the striatum one week post intrastriatal injection ofDi-HTT, Di-HTT-Cy3, or two negative controls (aCSF or Di-NTC). FIG. 24Bdepicts a scatter dot plot showing Htt mRNA expression in the cortex oneweek post intrastriatal injection of Di-HTT, Di-HTT-Cy3, or two negativecontrols (aCSF or Di-NTC).

FIG. 25 depicts a scatter dot plot measuring Di-HTT-Cy3 levels in thestriatum and cortex. The plot shows that significant levels ofDi-HTT-Cy3 are still detectable two weeks post intrastriatal injection.

FIGS. 26A-26B show that Di-HTT-Cy3 effectively silences HTT mRNA andprotein expression in both the striatum and the cortex two weeks postintrastriatal injection. FIG. 26A depicts a scatter dot plot measuringHtt mRNA levels in the striatum and cortex two weeks post injection.FIG. 26B depicts a scatter dot plot measuring Htt protein levels in thestriatum and cortex two weeks post injection.

FIGS. 27A-27B show that high dose Di-HTT-Cy3 treatment does not causesignificant toxicity in vivo but does lead to significant gliosis invivo two weeks post intrastriatal injection. FIG. 27A depicts a scatterdot plot measuring DARPP32 signal in the striatum and cortex two weeksafter injection with Di-HTT-Cy3 or aCSF. FIG. 27B depicts a scatter dotplot measuring GFAP protein levels in the striatum and cortex two weeksafter injection with Di-HTT-Cy3 or aCSF.

FIG. 28 depicts fluorescent imaging showing that intrathecal injectionof Di-HTT-Cy3 results in robust and even distribution throughout thespinal cord.

FIG. 29 depicts a merged fluorescent image of FIG. 28B (zoom of spinalcord). Blue-nuclei, red-Di-HTT-Cy3.XXX.

FIGS. 30A-30C show the widespread distribution of Di-HTT-Cy3 48 hourspost intracerebroventricular injection. FIG. 30A depicts fluorescentimaging of sections of the striatum, cortex, and cerebellum. FIG. 30Bdepicts brightfield images of the whole brain injected with control(aCSF) or Di-HTT-Cy3. FIG. 30C depicts a fluorescent image of a wholebrain section 48 hours after Di-HTT-Cy3 injection.

FIG. 31 shows that Di-HTT-Cy3 accumulates in multiple brain regions twoweeks post intracerebroventricular injection. A scatter dot plotmeasures the level of Di-HTT-Cy3 in multiple areas of the brain.

FIG. 32A shows that Di-HTT-Cy3 induces Htt gene silencing in multipleregions of the brain two weeks post intracerebroventricular injectioncompared to a negative control injection (aCSF). A scatter dot plotmeasures Htt mRNA levels in multiple areas of the brain. FIG. 32B showsthat Di-HTT-Cy3 induces Htt silencing in multiple regions of the braintwo weeks post intracerebroventricular injection compared to a negativecontrol injection (aCSF). A scatter dot plot measures Htt protein levelsin multiple areas of the brain.

FIG. 33 shows that intracerebroventricular injection of high doseDi-HTT-Cy3 causes minor toxicity in vivo. A scatter dot plot measuresDARPP32 signal in multiple regions of the brain following Di-HTT-Cy3 ofaCSF injection.

FIG. 34 shows that intracerebroventricular injection of high doseDi-HTT-Cy3 causes significant gliosis in vivo. A scatter dot plotmeasures DARPP32 signal in multiple regions of the brain followingDi-HTT-Cy3 of aCSF injection.

FIG. 35 shows that Di-HTT-Cy3 is distributed to multiple organsfollowing intravenous injection. Fluorescent images depict Di-HTT-Cy3levels in the heart, kidney, adrenal gland, and spleen followingintravenous injection of Di-HTT-Cy3 or a negative control (PBS).

FIG. 36 shows that Di-HTT-Cy3 accumulates in multiple organs followingintravenous injection. A scatter dot plot measures the levels ofDi-HTT-Cy3 in multiple tissues.

FIG. 37 illustrates the structures of hsiRNA and fully metabolized (FM)hsiRNA.

FIGS. 38A-38B show that full metabolic stabilization of hsiRNAs resultsin more efficacious gene silencing following intrastriatal injection ofhsiRNA^(HTT) or FM-hsiRNA^(HTT). FIG. 38A depicts a scatter dot plotmeasuring HTT mRNA levels up to 12 days after intrastriatal injection.FIG. 38B depicts a scatter dot plot measuring HTT mRNA levels up to 28days after intrastriatal injection.

FIG. 39 depicts the chemical diversity of single stranded fully modifiedoligonucleotides. The single stranded oligonucleotides can consist ofgapmers, mixmers, miRNA inhibitors, SSOs, PMOs, or PNAs.

FIG. 40 depicts Di-HTT with a TEG phosphoramidate linker. FIG. 40discloses SEQ ID NOS 5, 2, 5 and 2, respectively, in order ofappearance.

FIG. 41 depicts Di-HTT with a TEG di-phosphate linker. FIG. 41 disclosesSEQ ID NOS 5, 2, 5 and 2, respectively, in order of appearance.

FIG. 42 depicts variations of Di-HTT with either two oligonucleotidebranches or four oligonucleotide branches. FIG. 42 discloses SEQ ID NOS5 and 2, respectively, in order of appearance.

FIG. 43 depicts another variant of Di-HTT of a structure with twooligonucleotide branches and R2 attached to the linker. FIG. 43discloses SEQ ID NOS 5, 2, 5 and 2, respectively, in order ofappearance.

FIG. 44 depicts a first strategy for the incorporation of a hydrophobicmoeity into the branched oligonucleotide structures.

FIG. 45 depicts a second strategy for the incorporation of a hydrophobicmoeity into the branched oligonucleotide structures.

FIG. 46 depicts a third strategy for the incorporation of a hydrophobicmoeity into the branched oligonucleotide structures.

DETAILED DESCRIPTION

The present disclosure provides branched oligonucleotides (“compounds ofthe invention”) exhibiting unexpected improvement in distribution, invivo efficacy and safety. The branched oligonucleotides described hereinefficiently and stably delivered small RNAs to multiple regions of thebrain and multiple other organs, demonstrating unprecedented efficacy ofdelivery that has not been previously demonstrated with unconjugatedsmall RNAs.

The compositions described herein allow efficient, stable delivery ofsiRNA in order to promote potent silencing of therapeutic target genes.The compositions exhibit therapeutic potential for many hard to treatdiseases and overcome present challenges in employing RNA therapeutics.

In a first aspect, provided herein is a branched oligonucleotidecompound comprising two or more nucleic acids, wherein the nucleic acidsare connected to one another by one or more moieties selected from alinker, a spacer and a branching point.

Provided herein in various aspects and embodiments are branchedoligonucleotides, referred to as compounds of the invention. In someembodiments, compounds of the invention have two to eightoligonucleotides attached through a linker. The linker may behydrophobic. In a particular embodiment, compounds of the invention havetwo to three oligonucleotides. In one embodiment, the oligonucleotidesindependently have substantial chemical stabilization (e.g., at least40% of the constituent bases are chemically-modified). In a particularembodiment, the oliogonucleotides have full chemical stabilization(i.e., all of the constituent bases are chemically-modified). In someembodiments, compounds of the invention comprise one or moresingle-stranded phosphorothioated tails, each independently having twoto twenty nucleotides. In a particular embodiment, each single-strandedtail has eight to ten nucleotides.

In certain embodiments, compounds of the invention are characterized bythree properties: (1) a branched structure, (2) full metabolicstabilization, and (3) the presence of a single-stranded tail comprisingphosphorothioate linkers. In a particular embodiment, compounds of theinvention have 2 or 3 branches. The increased overall size of thebranched structures promote increased uptake. Also, without being boundby a particular theory of activity, multiple adjacent branches (e.g., 2or 3) allow each branch to act cooperatively and thus dramaticallyenhance rates of internalization, trafficking and release.

Full metabolic stabilization of branched oligonucleotides of theinvention results in unexpectedly high in vivo efficacy. Unstabilizedbranched siRNA lacks an in vivo efficacy. The presence of a singlestranded tail is required for the activity of branched oligonucleotides.The phosphoroamidate functional group is crucial for the function of thedi-branched oligos.

In certain embodiments, compounds of the invention are characterized bythe following properties: (1) two or more branched oligonucleotides,e.g., wherein there is a non-equal number of 3′ and 5′ ends; (2)substantially chemically stabilized, e.g., wherein more than 40%,optimally 100%, of oligonucleotides are chemically modified (e.g., noRNA and optionally no DNA); and (3) phoshorothioated singleoligonucleotides containing at least 3, optimally 5-20 phosphorothioatedbonds.

Compounds of the invention are provided in various structurally diverseembodiments. As shown in FIG. 7, for example, in some embodimentsoligonucleotides attached at the branching points are single strandedand consist of miRNA inhibitors, gapmers, mixmers, SSOs, PMOs, or PNAs.These single strands can be attached at their 3′ or 5′ end. Combinationsof siRNA and single stranded oligonucleotides could also be used fordual function. In another embodiment, short oligonucleotidescomplementary to the gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, andPNAs are used to carry these active single-stranded oligonucleotides andenhance distribution and cellular internalization. The short duplexregion has a low melting temperature (T_(m)˜37° C.) for fastdissociation upon internalization of the branched structure into thecell.

As shown in FIG. 16, Di-siRNA compounds of the invention may comprisechemically diverse conjugates. Conjugated bioactive ligands may be usedto enhance cellular specificity and to promote membrane association,internalization, and serum protein binding. Examples of bioactivemoieties to be used for conjugation include DHAg2, DHA, GalNAc, andcholesterol. These moieties can be attached to Di-siRNA either throughthe connecting linker or spacer, or added via an additional linker orspacer attached to another free siRNA end.

The presence of a branched structure improves the level of tissueretention in the brain more than 100-fold compared to non-branchedcompounds of identical chemical composition, suggesting a new mechanismof cellular retention and distribution. Compounds of the invention haveunexpectedly uniform distribution throughout the spinal cord and brain.Moreover, compounds of the invention exhibit unexpectedly efficientsystemic delivery to a variety of tissues, and very high levels oftissue accumulation.

Compounds of the invention comprise a variety of therapeuticoligonucleotides, including ASOs, miRNAs, miRNA inhibitors, spliceswitching, PMOs, PNAs. In some embodiments, compounds of the inventionfurther comprise conjugated hydrophobic moieties and exhibitunprecedented silencing and efficacy in vitro and in vivo.

Non-limiting embodiments of branched oligonucleotide configurations aredisclosed in FIGS. 1, 7-9, 15-17, and 40-45. Non-limiting examples oflinkers, spacers and branching points are disclosed in FIG. 6.

Variable Nucleic Acids

In an embodiment, the branched oligonucleotide comprises 2, 3, 4, 6 or 8nucleic acids. In one embodiment, the branched oligonucleotide comprises2 nucleic acids. In another embodiment, the branched oligonucleotidecomprises 3 nucleic acids. In another embodiment, the branchedoligonucleotide comprises 4 nucleic acids. In another embodiment, thebranched oligonucleotide comprises 6 nucleic acids. In anotherembodiment, the branched oligonucleotide comprises 8 nucleic acids. Inanother embodiment, the branched oligonucleotide comprises 5 nucleicacids. In another embodiment, the branched oligonucleotide comprises 7nucleic acids.

In an embodiment of the branched oligonucleotide, each nucleic acid issingle-stranded and has a 5′ end and a 3′ end, and each nucleic acid isindependently connected to a linker, a spacer, or a branching point atthe 5′ end or at the 3′ end. In one embodiment, each nucleic acid isconnected to a linker, spacer or branching point at the 3′ end. Inanother embodiment, each nucleic acid is connected to a linker, spaceror branching point at the 5′ end. In one embodiment, each nucleic acidis connected to a linker. In another embodiment, each nucleic acid isconnected to a spacer. In another embodiment, each nucleic acid isconnected to a branch point.

In an embodiment, each single-stranded nucleic acid independentlycomprises at least 15 contiguous nucleotides. In an embodiment, thenucleic acid comprises at least 15, at least 16, at least 17, at least18, at least 19, or at least 20 contiguous nucleotides, and hascomplementarity to a target. In certain embodiments, the complementarityis >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50%. In oneembodiment, the nucleic acid has perfect complementarity to a target.

In an embodiment of the branched oligonucleotide, each nucleic acid isdouble-stranded and comprises a sense strand and an antisense strand,wherein the sense strand and the antisense strand each have a 5′ end anda 3′ end. In an embodiment, each double-stranded nucleic acid isindependently connected to a linker, spacer or branching point at the 3′end or at the 5′ end of the sense strand or the antisense strand. In oneembodiment, each nucleic acid is connected to a linker, spacer orbranching point at the 3′ end of the sense strand. In anotherembodiment, each nucleic acid is connected to a linker, spacer orbranching point at the 3′ end of the antisense strand. In anotherembodiment, each nucleic acid is connected to a linker, spacer orbranching point at the 5′ end of the sense strand. In anotherembodiment, each nucleic acid is connected to a linker, spacer orbranching point at the 5′ end of the antisense strand. In oneembodiment, each nucleic acid is connected to a linker. In anotherembodiment, each nucleic acid is connected to a spacer. In anotherembodiment, each nucleic acid is connected to a branch point.

In an embodiment, each double-stranded nucleic acid independentlycomprises at least 15 contiguous nucleotides. In an embodiment, theantisense strand comprises at least 15, at least 16, at least 17, atleast 18, at least 19, or at least 20 contiguous nucleotides, and hascomplementarity to a target. In certain embodiments, the complementarityis >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50%. In oneembodiment, the antisense strand has perfect complementarity to atarget.

Modified Nucleotides

In an embodiment, each nucleic acid comprises one or morechemically-modified nucleotides. In an embodiment, each nucleic acidconsists of chemically-modified nucleotides. In certainembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of each nucleic acid comprises chemically-modified nucleotides.

In an embodiment, the sense strand and the antisense strand eachcomprise one or more chemically-modified nucleotides. In an embodiment,the sense strand and the antisense strand each consist ofchemically-modified nucleotides. In an embodiment, the sense strand andthe antisense strand both comprise alternating 2′-methoxy-nucleotidesand 2′-fluoro-nucleotides. In an embodiment, the nucleotides atpositions 1 and 2 from the 5′ end of the sense and antisense strands areconnected to adjacent nucleotides via phosphorothioate linkages. In anembodiment, the nucleotides at positions 1-6 from the 3′ end, orpositions 1-7 from the 3′ end, are connected to adjacent nucleotides viaphosphorothioate linkages. In other embodiments, at least 5 nucleotidesare connected to adjacent nucleotides via phosphorothioate linkages.

In an embodiment of the branched oligonucleotide, each linker isindependently selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; wherein anycarbon or oxygen atom of the linker is optionally replaced with anitrogen atom, bears a hydroxyl substituent, or bears an oxosubstituent. In one embodiment, each linker is an ethylene glycol chain.In another embodiment, each linker is an alkyl chain. In anotherembodiment, each linker is a peptide. In another embodiment, each linkeris RNA. In another embodiment, each linker is DNA. In anotherembodiment, each linker is a phosphate. In another embodiment, eachlinker is a phosphonate. In another embodiment, each linker is aphosphoramidate. In another embodiment, each linker is an ester. Inanother embodiment, each linker is an amide. In another embodiment, eachlinker is a triazole. In another embodiment, each linker is a structureselected from the formulas of FIG. 7.

Compound of Formula (I)

In a second aspect, provided herein is a compound of formula (I):

L—(N)_(n)  (I)

wherein L is selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof, wherein formula(I) optionally further comprises one or more branch point B, and one ormore spacer S; wherein B is independently for each occurrence apolyvalent organic species or derivative thereof; S is independently foreach occurrence selected from an ethylene glycol chain, an alkyl chain,a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof;

N is an RNA duplex comprising a sense strand and an antisense strand,wherein the sense strand and antisense strand each independentlycomprise one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7or 8.

In an embodiment, the compound of formula (I) has a structure selectedfrom formulas (I-1)-(I-9) of Table 1.

TABLE 1 N—L—N (I-1) N—S—L—S—N (I-2)

(I-3)

(I-4)

(I-5)

(I-6)

(I-7)

(I-8)

(I-9)

In one embodiment, the compound of formula (I) is formula (I-1). Inanother embodiment, the compound of formula (I) is formula (I-2). Inanother embodiment, the compound of formula (I) is formula (I-3). Inanother embodiment, the compound of formula (I) is formula (I-4). Inanother embodiment, the compound of formula (I) is formula (I-5). Inanother embodiment, the compound of formula (I) is formula (I-6). Inanother embodiment, the compound of formula (I) is formula (I-7). Inanother embodiment, the compound of formula (I) is formula (I-8). Inanother embodiment, the compound of formula (I) is formula (I-9).

In an embodiment of the compound of formula (I), each linker isindependently selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; wherein anycarbon or oxygen atom of the linker is optionally replaced with anitrogen atom, bears a hydroxyl substituent, or bears an oxosubstituent. In one embodiment of the compound of formula (I), eachlinker is an ethylene glycol chain. In another embodiment, each linkeris an alkyl chain. In another embodiment of the compound of formula (I),each linker is a peptide. In another embodiment of the compound offormula (I), each linker is RNA. In another embodiment of the compoundof formula (I), each linker is DNA. In another embodiment of thecompound of formula (I), each linker is a phosphate. In anotherembodiment, each linker is a phosphonate. In another embodiment of thecompound of formula (I), each linker is a phosphoramidate. In anotherembodiment of the compound of formula (I), each linker is an ester. Inanother embodiment of the compound of formula (I), each linker is anamide. In another embodiment of the compound of formula (I), each linkeris a triazole. In another embodiment of the compound of formula (I),each linker is a structure selected from the formulas of FIG. 7.

In one embodiment of the compound of formula (I), B is a polyvalentorganic species. In another embodiment of the compound of formula (I), Bis a derivative of a polyvalent organic species. In one embodiment ofthe compound of formula (I), B is a triol or tetrol derivative. Inanother embodiment, B is a tri- or tetra-carboxylic acid derivative. Inanother embodiment, B is an amine derivative. In another embodiment, Bis a tri- or tetra-amine derivative. In another embodiment, B is anamino acid derivative. In another embodiment of the compound of formula(I), B is selected from the formulas of FIG. 6.

Polyvalent organic species are moieties comprising carbon and three ormore valencies (i.e., points of attachment with moieties such as S, L orN, as defined above). Non-limiting examples of polyvalent organicspecies include triols (e.g., glycerol, phloroglucinol, and the like),tetrols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, andthe like), tri-carboxylic acids (e.g., citric acid,1,3,5-cyclohexanetricarboxylic acid, trimesic acid, and the like),tetra-carboxylic acids (e.g., ethylenediaminetetraacetic acid,pyromellitic acid, and the like), tertiary amines (e.g.,tripropargylamine, triethanolamine, and the like), triamines (e.g.,diethylenetriamine and the like), tetramines, and species comprising acombination of hydroxyl, thiol, amino, and/or carboxyl moieties (e.g.,amino acids such as lysine, serine, cysteine, and the like).

In an embodiment of the compound of formula (I), each nucleic acidcomprises one or more chemically-modified nucleotides. In an embodimentof the compound of formula (I), each nucleic acid consists ofchemically-modified nucleotides. In certain embodiments of the compoundof formula (I), >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of each nucleic acid comprises chemically-modified nucleotides.

In an embodiment, each antisense strand independently comprises a 5′terminal group R selected from the groups of Table 2.

TABLE 2

R¹

R²

R³

R⁴

R⁵

R⁶

R⁷

R⁸

In one embodiment, R is R₁. In another embodiment, R is R₂. In anotherembodiment, R is R₃. In another embodiment, R is R₄. In anotherembodiment, R is R₅. In another embodiment, R is R₆. In anotherembodiment, R is R₇. In another embodiment, R is R₈.

Structure of Formula (II)

In an embodiment, the compound of formula (I) the structure of formula(II):

wherein X, for each occurrence, independently, is selected fromadenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; Y, for each occurrence, independently, is selectedfrom adenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; — represents a phosphodiester internucleosidelinkage; ═ represents a phosphorothioate internucleoside linkage; and ≡represents, individually for each occurrence, a base-pairing interactionor a mismatch.

In certain embodiments, the structure of formula (II) does not containmismatches. In one embodiment, the structure of formula (II) contains 1mismatch. In another embodiment, the compound of formula (II) contains 2mismatches. In another embodiment, the compound of formula (II) contains3 mismatches. In another embodiment, the compound of formula (II)contains 4 mismatches. In an embodiment, each nucleic acid consists ofchemically-modified nucleotides.

In certainembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of formula (II) are chemically-modifiednucleotides. In otherembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of formula (II) are chemically-modifiednucleotides.

Structure of Formula (III)

In an embodiment, the compound of formula (I) has the structure offormula (III):

wherein X, for each occurrence, independently, is a nucleotidecomprising a 2′-deoxy-2′-fluoro modification; X, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification; Y,for each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; and Y, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification.

In an embodiment, X is chosen from the group consisting of2′-deoxy-2′-fluoro modified adenosine, guanosine, uridine or cytidine.In an embodiment, X is chosen from the group consisting of 2′-O-methylmodified adenosine, guanosine, uridine or cytidine. In an embodiment, Yis chosen from the group consisting of 2′-deoxy-2′-fluoro modifiedadenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosenfrom the group consisting of 2′-O-methyl modified adenosine, guanosine,uridine or cytidine.

In certain embodiments, the structure of formula (III) does not containmismatches. In one embodiment, the structure of formula (III) contains 1mismatch. In another embodiment, the compound of formula (III) contains2 mismatches. In another embodiment, the compound of formula (III)contains 3 mismatches. In another embodiment, the compound of formula(III) contains 4 mismatches.

Structure of Formula (IV)

In an embodiment, the compound of formula (I) has the structure offormula (IV) (SEQ ID NOS 1 and 2, respectively, in order of appearance):

wherein A is an adenosine comprising a 2′-deoxy-2′-fluoro modification;A is an adenosine comprising a 2′-O-methyl modification; G is anguanosine comprising a 2′-deoxy-2′-fluoro modification; G is anguanosine comprising a 2′-O-methyl modification; U is an uridinecomprising a 2′-deoxy-2′-fluoro modification; U is an uridine comprisinga 2′-O-methyl modification; C is an cytidine comprising a2′-deoxy-2′-fluoro modification; and C is an cytidine comprising a2′-O-methyl modification.

Structure of Formula (V)

In an embodiment, the compound of formula (I) has the structure offormula (V):

wherein X, for each occurrence, independently, is selected fromadenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; Y, for each occurrence, independently, is selectedfrom adenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; — represents a phosphodiester internucleosidelinkage; ═ represents a phosphorothioate internucleoside linkage; and ≡represents, individually for each occurrence, a base-pairing interactionor a mismatch.

In certain embodiments, the structure of formula (V) does not containmismatches. In one embodiment, the structure of formula (V) contains 1mismatch. In another embodiment, the compound of formula (V) contains 2mismatches. In another embodiment, the compound of formula (V) contains3 mismatches. In another embodiment, the compound of formula (V)contains 4 mismatches. In an embodiment, each nucleic acid consists ofchemically-modified nucleotides.

In certainembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of formula (II) are chemically-modifiednucleotides. In otherembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of formula (II) are chemically-modifiednucleotides.

Structure of Formula (VI)

In an embodiment, the compound of formula (I) has the structure offormula (VI):

wherein X, for each occurrence, independently, is a nucleotidecomprising a 2′-deoxy-2′-fluoro modification; X, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification; Y,for each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; and Y, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification.

In certain embodiments, X is chosen from the group consisting of2′-deoxy-2′-fluoro modified adenosine, guanosine, uridine or cytidine.In an embodiment, X is chosen from the group consisting of 2′-O-methylmodified adenosine, guanosine, uridine or cytidine. In an embodiment, Yis chosen from the group consisting of 2′-deoxy-2′-fluoro modifiedadenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosenfrom the group consisting of 2′-O-methyl modified adenosine, guanosine,uridine or cytidine.

In certain embodiments, the structure of formula (VI) does not containmismatches. In one embodiment, the structure of formula (VI) contains 1mismatch. In another embodiment, the compound of formula (VI) contains 2mismatches. In another embodiment, the compound of formula (VI) contains3 mismatches. In another embodiment, the compound of formula (VI)contains 4 mismatches.

Structure of Formula (VII)

In an embodiment, the compound of formula (I) has the structure offormula (VII) (SEQ ID NOS 3 and 4, respectively, in order ofappearance):

wherein A is an adenosine comprising a 2′-deoxy-2′-fluoro modification;A is an adenosine comprising a 2′-O-methyl modification; G is anguanosine comprising a 2′-deoxy-2′-fluoro modification; G is anguanosine comprising a 2′-O-methyl modification; U is an uridinecomprising a 2′-deoxy-2′-fluoro modification; U is an uridine comprisinga 2′-O-methyl modification; C is an cytidine comprising a2′-deoxy-2′-fluoro modification; C is an cytidine comprising a2′-O-methyl modification; Y, for each occurrence, independently, is anucleotide comprising a 2′-deoxy-2′-fluoro modification; and Y, for eachoccurrence, independently, is a nucleotide comprising a 2′-O-methylmodification.

Variable Linkers

In an embodiment of the compound of formula (I), L has the structure ofL1:

In an embodiment of L1, R is R³ and n is 2.

In an embodiment of the structure of formula (II), L has the structureof L1. In an embodiment of the structure of formula (III), L has thestructure of L1. In an embodiment of the structure of formula (IV), Lhas the structure of L1. In an embodiment of the structure of formula(V), L has the structure of L1. In an embodiment of the structure offormula (VI), L has the structure of L1. In an embodiment of thestructure of formula (VII), L has the structure of L1.

In an embodiment of the compound of formula (I), L has the structure ofL2:

In an embodiment of L2, R is R³ and n is 2.

In an embodiment of the structure of formula (II), L has the structureof L2. In an embodiment of the structure of formula (III), L has thestructure of L2. In an embodiment of the structure of formula (IV), Lhas the structure of L2. In an embodiment of the structure of formula(V), L has the structure of L2. In an embodiment of the structure offormula (VI), L has the structure of L2. In an embodiment of thestructure of formula (VII), L has the structure of L2.

Delivery System

In a third aspect, provided herein is a delivery system for therapeuticnucleic acids having the structure of formula (VIII):

L—(cNA)_(n)  (VIII)

wherein L is selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof, wherein formula(VIII) optionally further comprises one or more branch point B, and oneor more spacer S; wherein B is independently for each occurrence apolyvalent organic species or derivative thereof; S is independently foreach occurrence selected from an ethylene glycol chain, an alkyl chain,a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; each cNA,independently, is a carrier nucleic acid comprising one or more chemicalmodifications; and n is 2, 3, 4, 5, 6, 7 or 8.

In one embodiment of the delivery system, L is an ethylene glycol chain.In another embodiment of the delivery system, L is an alkyl chain. Inanother embodiment of the delivery system, L is a peptide. In anotherembodiment of the delivery system, L is RNA. In another embodiment ofthe delivery system, L is DNA. In another embodiment of the deliverysystem, L is a phosphate. In another embodiment of the delivery system,L is a phosphonate. In another embodiment of the delivery system, L is aphosphoramidate. In another embodiment of the delivery system, L is anester. In another embodiment of the delivery system, L is an amide. Inanother embodiment of the delivery system, L is a triazole.

In one embodiment of the delivery system, S is an ethylene glycol chain.In another embodiment, S is an alkyl chain. In another embodiment of thedelivery system, S is a peptide. In another embodiment, S is RNA. Inanother embodiment of the delivery system, S is DNA. In anotherembodiment of the delivery system, S is a phosphate. In anotherembodiment of the delivery system, S is a phosphonate. In anotherembodiment of the delivery system, S is a phosphoramidate. In anotherembodiment of the delivery system, S is an ester. In another embodiment,S is an amide. In another embodiment, S is a triazole.

In one embodiment of the delivery system, n is 2. In another embodimentof the delivery system, n is 3. In another embodiment of the deliverysystem, n is 4. In another embodiment of the delivery system, n is 5. Inanother embodiment of the delivery system, n is 6. In another embodimentof the delivery system, n is 7. In another embodiment of the deliverysystem, n is 8.

In certain embodiments, each cNAcomprises >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50%chemically-modified nucleotides.

In an embodiment, the compound of formula (VIII) has a structureselected from formulas (VIII-1)-(VIII-9) of Table 3:

TABLE 3 ANc—L—cNA (VIII-1) ANc—S—L—S—cNA (VIII-2)

(VIII-3)

(VIII-4)

(VIII-5)

(VIII-6)

(VIII-7)

(VIII-8)

(VIII-9)

In an embodiment, the compound of formula (VIII) is the structure offormula (VIII-1). In an embodiment, the compound of formula (VIII) isthe structure of formula (VIII-2). In an embodiment, the compound offormula (VIII) is the structure of formula (VIII-3). In an embodiment,the compound of formula (VIII) is the structure of formula (VIII-4). Inan embodiment, the compound of formula (VIII) is the structure offormula (VIII-5). In an embodiment, the compound of formula (VIII) isthe structure of formula (VIII-6). In an embodiment, the compound offormula (VIII) is the structure of formula (VIII-7). In an embodiment,the compound of formula (VIII) is the structure of formula (VIII-8). Inan embodiment, the compound of formula (VIII) is the structure offormula (VIII-9).

In an embodiment, the compound of formulas (VIII) (including, e.g.,formulas (VIII-1)-(VIII-9), each cNA independently comprises at least 15contiguous nucleotides. In an embodiment, each cNA independentlyconsists of chemically-modified nucleotides.

In an embodiment, the delivery system further comprises n therapeuticnucleic acids (NA), wherein each NA is hybridized to at least one cNA.In one embodiment, the delivery system is comprised of 2 NAs. In anotherembodiment, the delivery system is comprised of 3 NAs. In anotherembodiment, the delivery system is comprised of 4 NAs. In anotherembodiment, the delivery system is comprised of 5 NAs. In anotherembodiment, the delivery system is comprised of 6 NAs. In anotherembodiment, the delivery system is comprised of 7 NAs. In anotherembodiment, the delivery system is comprised of 8 NAs.

In an embodiment, each NA independently comprises at least 16 contiguousnucleotides. In an embodiment, each NA independently comprises 16-20contiguous nucleotides. In an embodiment, each NA independentlycomprises 16 contiguous nucleotides. In another embodiment, each NAindependently comprises 17 contiguous nucleotides. In anotherembodiment, each NA independently comprises 18 contiguous nucleotides.In another embodiment, each NA independently comprises 19 contiguousnucleotides. In another embodiment, each NA independently comprises 20contiguous nucleotides.

In an embodiment, each NA comprises an unpaired overhang of at least 2nucleotides. In another embodiment, each NA comprises an unpairedoverhang of at least 3 nucleotides. In another embodiment, each NAcomprises an unpaired overhang of at least 4 nucleotides. In anotherembodiment, each NA comprises an unpaired overhang of at least 5nucleotides. In another embodiment, each NA comprises an unpairedoverhang of at least 6 nucleotides. In an embodiment, the nucleotides ofthe overhang are connected via phosphorothioate linkages.

In an embodiment, each NA, independently, is selected from the groupconsisting of: DNA, siRNAs, antagomiRs, miRNAs, gapmers, mixmers, orguide RNAs. In one embodiment, each NA, independently, is a DNA. Inanother embodiment, each NA, independently, is a siRNA. In anotherembodiment, each NA, independently, is an antagomiR. In anotherembodiment, each NA, independently, is a miRNA. In another embodiment,each NA, independently, is a gapmer. In another embodiment, each NA,independently, is a mixmer. In another embodiment, each NA,independently, is a guide RNA. In an embodiment, each NA is the same. Inan embodiment, each NA is not the same.

In an embodiment, the delivery system further comprising n therapeuticnucleic acids (NA) has a structure selected from formulas (I), (II),(III), (IV), (V), (VI), (VII), and embodiments thereof described herein.In one embodiment, the delivery system has a structure selected fromformulas (I), (II), (III), (IV), (V), (VI), (VII), and embodimentsthereof described herein further comprising 2 therapeutic nucleic acids(NA). In another embodiment, the delivery system has a structureselected from formulas (I), (II), (III), (IV), (V), (VI), (VII), andembodiments thereof described herein further comprising 3 therapeuticnucleic acids (NA). In one embodiment, the delivery system has astructure selected from formulas (I), (II), (III), (IV), (V), (VI),(VII), and embodiments thereof described herein further comprising 4therapeutic nucleic acids (NA). In one embodiment, the delivery systemhas a structure selected from formulas (I), (II), (III), (IV), (V),(VI), (VII), and embodiments thereof described herein further comprising5 therapeutic nucleic acids (NA). In one embodiment, the delivery systemhas a structure selected from formulas (I), (II), (III), (IV), (V),(VI), (VII), and embodiments thereof described herein further comprising6 therapeutic nucleic acids (NA). In one embodiment, the delivery systemhas a structure selected from formulas (I), (II), (III), (IV), (V),(VI), (VII), and embodiments thereof described herein further comprising7 therapeutic nucleic acids (NA). In one embodiment, the delivery systemhas a structure selected from formulas (I), (II), (III), (IV), (V),(VI), (VII), and embodiments thereof described herein further comprising8 therapeutic nucleic acids (NA).

In one embodiment, the delivery system has a structure selected fromformulas (I), (II), (III), (IV), (V), (VI), (VII) further comprising alinker of structure L1 or L2 wherein R is R³ and n is 2. In anotherembodiment, the delivery system has a structure selected from formulas(I), (II), (III), (IV), (V), (VI), (VII) further comprising a linker ofstructure L1 wherein R is R³ and n is 2. In another embodiment, thedelivery system has a structure selected from formulas (I), (II), (III),(IV), (V), (VI), (VII) further comprising a linker of structure L2wherein R is R³ and n is 2.

In an embodiment of the delivery system, the target of delivery isselected from the group consisting of: brain, liver, skin, kidney,spleen, pancreas, colon, fat, lung, muscle, and thymus. In oneembodiment, the target of delivery is the brain. In another embodiment,the target of delivery is the striatum of the brain. In anotherembodiment, the target of delivery is the cortex of the brain. Inanother embodiment, the target of delivery is the striatum of the brain.In one embodiment, the target of delivery is the liver. In oneembodiment, the target of delivery is the skin. In one embodiment, thetarget of delivery is the kidney. In one embodiment, the target ofdelivery is the spleen. In one embodiment, the target of delivery is thepancreas. In one embodiment, the target of delivery is the colon. In oneembodiment, the target of delivery is the fat. In one embodiment, thetarget of delivery is the lung. In one embodiment, the target ofdelivery is the muscle. In one embodiment, the target of delivery is thethymus. In one embodiment, the target of delivery is the spinal cord.

It is to be understood that the methods described in this disclosure arenot limited to particular methods and experimental conditions disclosedherein; as such methods and conditions may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Furthermore, the experiments described herein, unless otherwiseindicated, use conventional molecular and cellular biological andimmunological techniques within the skill of the art. Such techniquesare well known to the skilled worker, and are explained fully in theliterature. See, e.g., Ausubel, et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., NY (1987-2008), includingall supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition)by M R Green and J. Sambrook and Harlow et al., Antibodies: A LaboratoryManual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor(2013, 2nd edition).

Definitions

Unless otherwise defined herein, scientific and technical terms usedherein have the meanings that are commonly understood by those ofordinary skill in the art. In the event of any latent ambiguity,definitions provided herein take precedent over any dictionary orextrinsic definition. Unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including,” as well as other forms, such as “includes”and “included,” is not limiting.

As used herein, the term “nucleic acids” refers to RNA or DNA moleculesconsisting of a chain of ribonucleotides or deoxyribonucleotides,respectively.

As used herein, the term “therapeutic nucleic acid” refers to a nucleicacid molecule (e.g., ribonucleic acid) that has partial or completecomplementarity to, and interacts with, a disease-associated target mRNAand mediates silencing of expression of the mRNA.

As used herein, the term “carrier nucleic acid” refers to a nucleic acidmolecule (e.g., ribonucleic acid) that has sequence complementaritywith, and hybridizes with, a therapeutic nucleic acid.

As used herein, the term “3′ end” refers to the end of the nucleic acidthat contains an unmodified hydroxyl group at the 3′ carbon of theribose ring.

As used herein, the term “5′ end” refers to the end of the nucleic acidthat contains a phosphate group attached to the 5′ carbon of the ribosering.

As used herein, the term “nucleoside” refers to a molecule made up of aheterocyclic base and its sugar.

As used herein, the term “nucleotide” refers to a nucleoside having aphosphate group on its 3′ or 5′ sugar hydroxyl group.

As used herein, the term “siRNA” refers to small interfering RNAduplexes that induce the RNA interference (RNAi) pathway. siRNAmolecules can vary in length (generally between 18-30 basepairs) andcontain varying degrees of complementarity to their target mRNA. Theterm “siRNA” includes duplexes of two separate strands, as well assingle strands that can form hairpin structures comprising a duplexregion.

As used herein, the term “antisense strand” refers to the strand of thesiRNA duplex that contains some degree of complementarity to the targetgene.

As used herein, the term “sense strand” refers to the strand of thesiRNA duplex that contains complementarity to the antisense strand.

As used herein, the terms “chemically modified nucleotide” or“nucleotide analog” or “altered nucleotide” or “modified nucleotide”refer to a non-standard nucleotide, including non-naturally occurringribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogsare modified at any position so as to alter certain chemical propertiesof the nucleotide yet retain the ability of the nucleotide analog toperform its intended function. Examples of positions of the nucleotidewhich may be derivatized include the 5 position, e.g., 5-(2-amino)propyluridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.;the 6 position, e.g., 6-(2-amino)propyl uridine; the 8-position foradenosine and/or guanosines, e.g., 8-bromo guanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g.,alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art)nucleotides; and other heterocyclically modified nucleotide analogs suchas those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portionof the nucleotides. For example the 2′ OH-group may be replaced by agroup selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR₂, COOR,or OR, wherein R is substituted or unsubstituted C1-C6 alkyl, alkenyl,alkynyl, aryl, etc. Other possible modifications include those describedin U.S. Pat. Nos. 5,858,988, and 6,291,438.

As used herein, the term “metabolically stabilized” refers to RNAmolecules that contain ribonucleotides that have been chemicallymodified from 2′-hydroxyl groups to 2′-O-methyl groups.

As used herein, the term “phosphorothioate” refers to the phosphategroup of a nucleotide that is modified by substituting one or more ofthe oxygens of the phosphate group with sulfur.

As used herein, the term “ethylene glycol chain” refers to a carbonchain with the formula ((CH₂OH)₂).

As used herein, the term “alkyl chain” refers to an acyclic unsaturatedhydrocarbon chain. In connection with this invention an “alkyl chain”includes but is not limited to straight chain, branch chain, and cyclicunsaturated hydrocarbon groups.

As used herein, the term “amide” refers to an alkyl or aromatic groupthat is attached to an amino-carbonyl functional group.

As used herein, the term “internucleoside” and “internucleotide” referto the bonds between nucleosides and nucleotides, respectively.

As used herein, the term “triazol” refers to heterocyclic compounds withthe formula (C₂H₃N₃), having a five-membered ring of two carbons andthree nitrogens, the positions of which can change resulting in multipleisomers.

As used herein, the term “terminal group” refers to the group at which acarbon chain or nucleic acid ends.

As used herein, the term “lipophilic amino acid” refers to an amino acidcomprising a hydrophobic moiety (e.g., an alkyl chain or an aromaticring).

As used herein, the term “antagomiRs” refers to nucleic acids that canfunction as inhibitors of miRNA activity.

As used herein, the term “gapmers” refers to chimeric antisense nucleicacids that contain a central block of deoxynucleotide monomerssufficiently long to induce RNase H cleavage. The deoxynucleotide blockis flanked by ribonucleotide monomers or ribonucleotide monomerscontaining modifications.

As used herein, the term “mixmers” refers to nucleic acids that arecomprised of a mix of locked nucleic acids (LNAs) and DNA.

As used herein, the term “guide RNAs” refers to refers to nucleic acidsthat have sequence complementarity to a specific sequence in the genomeimmediately or 1 base pair upstream of the protospacer adjacent motif(PAM) sequence as used in CRISPR/Cas9 gene editing systems.

As used herein, the term “target of delivery” refers to the organ orpart of the body that is desired to deliver the branched oligonucleotidecompositions to.

As used herein, the term “Di-siRNA” refers to a molecule of the presentinvention that comprises a branched oligonucleotide structure andcontains siRNA molecules as the therapeutic nucleic acids.

As used herein, an “amino acid” refers to a molecule containing amineand carboxyl functional groups and a side chain (R) specific to theamino acid. In one embodiment, an amino acid has a structure of theformula:

In another embodiment, “amino acid” may refer to a component residue ofa peptide or protein having a structure of the formula:

In some embodiments the amino acid is chosen from the group ofproteinogenic amino acids. In other embodiments, the amino acid is anL-amino acid or a D-amino acid. In other embodiments, the amino acid isa synthetic amino acid (e.g., a beta-amino acid).

It is understood that certain internucleotide linkages provided herein,including, e.g., phosphodiester and phosphorothioate, comprise a formalcharge of -1 at physiological pH, and that said formal charge will bebalanced by a cationic moiety, e.g., an alkali metal such as sodium orpotassium, an alkali earth metal such as calcium or magnesium, or anammonium or guanidinium ion.

The phosphate group of the nucleotide may also be modified, e.g., bysubstituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct.11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)preferably decrease the rate of hydrolysis of, for example,polynucleotides comprising said analogs in vivo or in vitro.

Delivery and Distribution

In another aspect, provided herein is a method for selectivelydelivering a nucleic acid as described herein to a particular organ in apatient, comprising administering to the patient a branchedoligonucleotide as described herein, such that the nucleic acid isdelivered selectively. In one embodiment, the organ is the liver. Inanother embodiment, the organ is the kidneys. In another embodiment, theorgan is the spleen. In another embodiment, the organ is the heart. Inanother embodiment, the organ is the brain. In another embodiment, thenucleic acid

The compositions described herein promote simple, efficient, non-toxicdelivery of metabolically stable oligonucleotides (e.g., siRNA), andpromote potent silencing of therapeutic targets in a range of tissues invivo.

As shown in FIG. 11, Di-siRNA distributes throughout the injectedhemisphere of the mouse brain following intrastriatal injection. While asingle non conjugated siRNA can silence mRNA in primary neurons, theDi-siRNA structure is essential for enhanced tissue distribution andtissue retention of modified oligo nucleotides. Other conjugates such ascholesterol, although retained, show a steep gradient of diffusion awayfrom the site of injection. The subtle hydrophobicity of the two singlestranded phosphorothioated tails supports tissue retention while alsoallowing for widespread and uniform distribution throughout theipsilateral hemisphere of the injected brain.

As shown in FIG. 12, a single injection of Di-siRNA was detected bothipsilateral and contralateral to the injection site, indicating thatspread is not limited to the injected hemisphere, but is also occurringacross the midline into the non-injected side. Alternative methods ofinjection, including intracerebral ventricular, may also facilitatebilateral distribution with only one injection.

Di-siRNA shows a very unique cellular distribution when injectedintrastriatally into the brain. Fluorescently labeled Di-siRNA appearsto localize preferentially with neurons in the cortex. This selectivefeature is specific to these compounds and is not true for other siRNAconjugates such as cholesterol which show no cell type preference.

Di-siRNA shows localization to fiber tracts in the striatum but ispresent within neuronal cell bodies in the cortex. Movement to thecortex may be through diffusion or may be the result of retrogradetransport via striatal fiber tracts. The theory that retrogradetransport is partially responsible is supported by the fact that someareas of the cortex show full neuronal penetration while neurons inadjacent areas show no Di-siRNA association.

A single therapeutically relevant brain injection of Di-siRNA results inwidespread distribution of Di-siRNA throughout the brain. The level ofdistribution demonstrated in FIGS. 31-32 is unprecedented in the priorart and shows that Di-siRNAs are a promising therapeutic deliverysystem.

Di-siRNA shows widespread distribution throughout the body following asingle intravenous injection. As shown in FIG. 37, significant levels ofDi-siRNAs were detected in mouse liver, skin, brain, kidney, spleen,pancreas, colon, fat, lung, muscle, and thymus. The finding thatDi-siRNAs are present in the brain following intravenous injection alsodemonstrates that the Di-siRNA structures efficiently cross theblood-brain barrier.

Silencing

In some embodiments, compounds of the invention promote about 90%striatal silencing and about 65% cortical silencing in vivo in brainwith a single injection, with no indication of toxicity. In someembodiments, compounds of the invention exhibit about 60% silencingthroughout all regions of the spinal cord with intrathecal injection.

Single injection of Di-siRNA induces robust silencing in both thestriatum and cortex of mouse brain. This level of efficacy has neverbeen demonstrated previously for non-conjugated siRNAs. AlthoughDi-siRNA appears visually associated with fiber tracts in striatum, theefficacy observed clearly indicates that striatal neurons areinternalizing Di-siRNA to a significant degree. In experiments,intrastriatal injection 2 nmols Di-siRNA (4 nmols of correspondingantisense HTT strand). Animals sacrificed 7 days post-injection. Tissuepunches taken from the 300 um brain slices from the striatum and cortex.Di-siRNA antisense strands present in different brain regions, liver,and kidney were quantified using Cy3-labeled complimentary PNA tohybridize to the strand and HPLC to quantify ng of oligo per mg oftissue. aCSF—Artificial CSF.

As shown in FIG. 10, Di-siRNA shows equal efficacy relative to a singlesiRNA duplex following lipid-mediated transfection in HeLa cells,indicating that RISC loading is not hindered by the tethering of twosiRNA duplexes to a linker. Di-siRNA is not efficacious in HeLa cellswithout transfection, however in primary cortical neurons, onephosphorothiated tail is enough to induce at least 60% silencing,suggesting that phosphorothiation is an effective method for deliveringsiRNA to primary neurons, without formulation.

As shown in FIG. 13, a single injection of Di-siRNA induces robustsilencing in both the striatum and cortex of mouse brain. Although a 63×image of pyrimidal neurons containing Cy3-labeled Di-branched oligoshows that Di-siRNA is visually associated with fiber tracts in thestriatum, the efficacy observed clearly indicates that striatal neuronsare internalizing Di-siRNA to a significant degree.

As shown in FIG. 14, Di-siRNA shows robust and even silencing throughoutthe spinal cord following intrathecal injection. A single injection ofDi-siRNA in the lumbar region of the spinal cord silences mRNA to thesame degree in the cervical, thoracic and lumbar regions indicating evenand long range distribution.

As shown in FIG. 24, Di-siRNA labeled with Cy3 induces robust silencingin both the striatum and cortex of mouse brain. The mRNA expressionlevels show that the addition of Cy3 to the branched oligonucleotidecompositions enhances silencing as compared to the unlabeled Di-siRNA.

As shown in FIGS. 23-24, a single intrastriatal injection resulted insilencing in both the cortex and striatum of mouse brain but did notresult in any significant silencing in the liver or kidney. Thisdemonstrates that branched oligonucleotides can specifically target anorgan of interest.

As shown in FIG. 26, a single injection of Di-siRNA continues tomaintain robust silencing two weeks after the injection in both thestriatum and cortex of mouse brain. Di-siRNA is stable and effective forat least two weeks in vivo.

As shown in FIG. 33 and FIG. 34, a therapeutically relevant singleinjection of Di-siRNA induces significant silencing in multiple areas ofthe brain. This is the first example of widespread siRNA silencing inthe brain following a single therapeutically relevant injection. Theseresults demonstrate that Di-siRNAs are an efficacious option for RNAtherapeutics.

Modified RNA Silencing Agents

In certain aspects of the invention, an RNA silencing agent (or anyportion thereof) of the invention as described supra may be modifiedsuch that the activity of the agent is further improved. For example,the RNA silencing agents described in above may be modified with any ofthe modifications described infra. The modifications can, in part, serveto further enhance target discrimination, to enhance stability of theagent (e.g., to prevent degradation), to promote cellular uptake, toenhance the target efficiency, to improve efficacy in binding (e.g., tothe targets), to improve patient tolerance to the agent, and/or toreduce toxicity.

1) Modifications to Enhance Target Discrimination

In certain embodiments, the RNA silencing agents of the invention may besubstituted with a destabilizing nucleotide to enhance single nucleotidetarget discrimination (see U.S. application Ser. No. 11/698,689, filedJan. 25, 2007 and U.S. Provisional Application No. 60/762,225 filed Jan.25, 2006, both of which are incorporated herein by reference). Such amodification may be sufficient to abolish the specificity of the RNAsilencing agent for a non-target mRNA (e.g. wild-type mRNA), withoutappreciably affecting the specificity of the RNA silencing agent for atarget mRNA (e.g. gain-of-function mutant mRNA).

In preferred embodiments, the RNA silencing agents of the invention aremodified by the introduction of at least one universal nucleotide in theantisense strand thereof. Universal nucleotides comprise base portionsthat are capable of base pairing indiscriminately with any of the fourconventional nucleotide bases (e.g. A, G, C, U). A universal nucleotideis preferred because it has relatively minor effect on the stability ofthe RNA duplex or the duplex formed by the guide strand of the RNAsilencing agent and the target mRNA. Exemplary universal nucleotideinclude those having an inosine base portion or an inosine analog baseportion selected from the group consisting of deoxyinosine (e.g.2′-deoxyinosine), 7-deaza-2′-deoxyinosine, 2′-aza-2′-deoxyinosine,PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine,2′-O-methoxyethyl-inosine, and 2′-OMe-inosine. In particularly preferredembodiments, the universal nucleotide is an inosine residue or anaturally occurring analog thereof

In certain embodiments, the RNA silencing agents of the invention aremodified by the introduction of at least one destabilizing nucleotidewithin 5 nucleotides from a specificity-determining nucleotide (i.e.,the nucleotide which recognizes the disease-related polymorphism). Forexample, the destabilizing nucleotide may be introduced at a positionthat is within 5, 4, 3, 2, or 1 nucleotide(s) from aspecificity-determining nucleotide. In exemplary embodiments, thedestabilizing nucleotide is introduced at a position which is 3nucleotides from the specificity-determining nucleotide (i.e., such thatthere are 2 stabilizing nucleotides between the destablilizingnucleotide and the specificity-determining nucleotide). In RNA silencingagents having two strands or strand portions (e.g. siRNAs and shRNAs),the destabilizing nucleotide may be introduced in the strand or strandportion that does not contain the specificity-determining nucleotide. Inpreferred embodiments, the destabilizing nucleotide is introduced in thesame strand or strand portion that contains the specificity-determiningnucleotide.

2) Modifications to Enhance Efficacy and Specificity

In certain embodiments, the RNA silencing agents of the invention may bealtered to facilitate enhanced efficacy and specificity in mediatingRNAi according to asymmetry design rules (see U.S. Pat. Nos. 8,309,704,7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such alterationsfacilitate entry of the antisense strand of the siRNA (e.g., a siRNAdesigned using the methods of the invention or an siRNA produced from ashRNA) into RISC in favor of the sense strand, such that the antisensestrand preferentially guides cleavage or translational repression of atarget mRNA, and thus increasing or improving the efficiency of targetcleavage and silencing. Preferably the asymmetry of an RNA silencingagent is enhanced by lessening the base pair strength between theantisense strand 5′ end (AS 5′) and the sense strand 3′ end (S 3′) ofthe RNA silencing agent relative to the bond strength or base pairstrength between the antisense strand 3′ end (AS 3′) and the sensestrand 5′ end (S 5′) of said RNA silencing agent.

In one embodiment, the asymmetry of an RNA silencing agent of theinvention may be enhanced such that there are fewer G:C base pairsbetween the 5′ end of the first or antisense strand and the 3′ end ofthe sense strand portion than between the 3′ end of the first orantisense strand and the 5′ end of the sense strand portion. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one mismatched base pair betweenthe 5′ end of the first or antisense strand and the 3′ end of the sensestrand portion. Preferably, the mismatched base pair is selected fromthe group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one wobble base pair, e.g., G:U,between the 5′ end of the first or antisense strand and the 3′ end ofthe sense strand portion. In another embodiment, the asymmetry of an RNAsilencing agent of the invention may be enhanced such that there is atleast one base pair comprising a rare nucleotide, e.g., inosine (I).Preferably, the base pair is selected from the group consisting of anI:A, I:U and I:C. In yet another embodiment, the asymmetry of an RNAsilencing agent of the invention may be enhanced such that there is atleast one base pair comprising a modified nucleotide. In preferredembodiments, the modified nucleotide is selected from the groupconsisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

3) RNA Silencing Agents with Enhanced Stability

The RNA silencing agents of the present invention can be modified toimprove stability in serum or in growth medium for cell cultures. Inorder to enhance the stability, the 3′-residues may be stabilizedagainst degradation, e.g., they may be selected such that they consistof purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine by 2′-deoxythymidine istolerated and does not affect the efficiency of RNA interference.

In a preferred aspect, the invention features RNA silencing agents thatinclude first and second strands wherein the second strand and/or firststrand is modified by the substitution of internal nucleotides withmodified nucleotides, such that in vivo stability is enhanced ascompared to a corresponding unmodified RNA silencing agent. As definedherein, an “internal” nucleotide is one occurring at any position otherthan the 5′ end or 3′ end of nucleic acid molecule, polynucleotide oroligonucleotide. An internal nucleotide can be within a single-strandedmolecule or within a strand of a duplex or double-stranded molecule. Inone embodiment, the sense strand and/or antisense strand is modified bythe substitution of at least one internal nucleotide. In anotherembodiment, the sense strand and/or antisense strand is modified by thesubstitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. Inanother embodiment, the sense strand and/or antisense strand is modifiedby the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of theinternal nucleotides. In yet another embodiment, the sense strand and/orantisense strand is modified by the substitution of all of the internalnucleotides.

In a preferred embodiment of the present invention, the RNA silencingagents may contain at least one modified nucleotide analogue. Thenucleotide analogues may be located at positions where thetarget-specific silencing activity, e.g., the RNAi mediating activity ortranslational repression activity is not substantially effected, e.g.,in a region at the 5′-end and/or the 3′-end of the siRNA molecule.Particularly, the ends may be stabilized by incorporating modifiednucleotide analogues.

Exemplary nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In exemplary backbone-modified ribonucleotides, the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In exemplary sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

In particular embodiments, the modifications are 2′-fluoro, 2′-aminoand/or 2′-thio modifications. Particularly preferred modificationsinclude 2′-fluoro-cytidine, 2′-fluorouridine, 2′-fluoro-adenosine,2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine,2′-amino-adenosine, 2′-amino-guanosine, 2,6-diaminopurine,4-thio-uridine, and/or 5-amino-allyl-uridine. In a particularembodiment, the 2′-fluoro ribonucleotides are every uridine andcytidine. Additional exemplary modifications include 5-bromo-uridine,5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine,2′-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and5-fluoro-uridine. 2′-deoxy-nucleotides and 2′-Ome nucleotides can alsobe used within modified RNA-silencing agents moities of the instantinvention. Additional modified residues include, deoxy-abasic, inosine,N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purineribonucleoside and ribavirin. In a particularly preferred embodiment,the 2′ moiety is a methyl group such that the linking moiety is a2-O-methyl oligonucleotide.

In an exemplary embodiment, the RNA silencing agent of the inventioncomprises Locked Nucleic Acids (LNAs). LNAs comprise sugar-modifiednucleotides that resist nuclease activities (are highly stable) andpossess single nucleotide discrimination for mRNA (Elmen et al., NucleicAcids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). Thesemolecules have 2′-O,4′-C-ethylene-bridged nucleic acids, with possiblemodifications such as 2′-deoxy-2″-fluorouridine. Moreover, LNAs increasethe specificity of oligonucleotides by constraining the sugar moietyinto the 3′-endo conformation, thereby pre-organizing the nucleotide forbase pairing and increasing the melting temperature of theoligonucleotide by as much as 10° C. per base.

In another exemplary embodiment, the RNA silencing agent of theinvention comprises Peptide Nucleic Acids (PNAs). PNAs comprise modifiednucleotides in which the sugar-phosphate portion of the nucleotide isreplaced with a neutral 2-amino ethylglycine moiety capable of forming apolyamide backbone which is highly resistant to nuclease digestion andimparts improved binding specificity to the molecule (Nielsen, et al.,Science, (2001), 254: 1497-1500).

Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

In other embodiments, cross-linking can be employed to alter thepharmacokinetics of the RNA silencing agent, for example, to increasehalf-life in the body. Thus, the invention includes RNA silencing agentshaving two complementary strands of nucleic acid, wherein the twostrands are crosslinked. The invention also includes RNA silencingagents which are conjugated or unconjugated (e.g., at its 3′ terminus)to another moiety (e.g. a non-nucleic acid moiety such as a peptide), anorganic compound (e.g., a dye), or the like). Modifying siRNAderivatives in this way may improve cellular uptake or enhance cellulartargeting activities of the resulting siRNA derivative as compared tothe corresponding siRNA, are useful for tracing the siRNA derivative inthe cell, or improve the stability of the siRNA derivative compared tothe corresponding siRNA.

Other exemplary modifications include: (a) 2′ modification, e.g.,provision of a 2′ OMe moiety on a U in a sense or antisense strand, butespecially on a sense strand, or provision of a 2′ OMe moiety in a 3′overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom ofthe molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′position, as indicated by the context); (b) modification of thebackbone, e.g., with the replacement of an 0 with an S, in the phosphatebackbone, e.g., the provision of a phosphorothioate modification, on theU or the A or both, especially on an antisense strand; e.g., with thereplacement of a P with an S; (c) replacement of the U with a C5 aminolinker; (d) replacement of an A with a G (sequence changes are preferredto be located on the sense strand and not the antisense strand); and (d)modification at the 2′, 6′, 7′, or 8′ position. Exemplary embodimentsare those in which one or more of these modifications are present on thesense but not the antisense strand, or embodiments where the antisensestrand has fewer of such modifications. Yet other exemplarymodifications include the use of a methylated P in a 3′ overhang, e.g.,at the 3′ terminus; combination of a 2′ modification, e.g., provision ofa 2′ OMe moiety and modification of the backbone, e.g., with thereplacement of a P with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P, in a 3′ overhang, e.g., atthe 3′ terminus; modification with a 3′ alkyl; modification with anabasic pyrrolidone in a 3′ overhang, e.g., at the 3′ terminus;modification with naproxen, ibuprofen, or other moieties which inhibitdegradation at the 3′ terminus.

4) Modifications to Enhance Cellular Uptake

In other embodiments, a compound of the invention may be modified withchemical moieties, for example, to enhance cellular uptake by targetcells (e.g., neuronal cells). Thus, the invention includes RNA silencingagents which are conjugated or unconjugated (e.g., at its 3′ terminus)to another moiety (e.g. a non-nucleic acid moiety such as a peptide), anorganic compound (e.g., a dye), or the like. The conjugation can beaccomplished by methods known in the art, e.g., using the methods ofLambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describesnucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles);Fattal et al., J. Control Release 53(1-3):137-43 (1998) (describesnucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalatingagents, hydrophobic groups, polycations or PACA nanoparticles); andGodard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleicacids linked to nanoparticles).

In a particular embodiment, a compound of the invention is conjugated toa lipophilic moiety. In one embodiment, the lipophilic moiety is aligand that includes a cationic group. In another embodiment, thelipophilic moiety is attached to one or both strands of an siRNA. In anexemplary embodiment, the lipophilic moiety is attached to one end ofthe sense strand of the siRNA. In another exemplary embodiment, thelipophilic moiety is attached to the 3′ end of the sense strand. Incertain embodiments, the lipophilic moiety is selected from the groupconsisting of cholesterol, vitamin D, DHA, DHAg2, EPA, vitamin E,vitamin K, vitamin A, folic acid, or a cationic dye (e.g., Cy3).

5) Tethered Ligands

Other entities can be tethered to a compound of the invention. Forexample, a ligand tethered to an RNA silencing agent to improvestability, hybridization thermodynamics with a target nucleic acid,targeting to a particular tissue or cell-type, or cell permeability,e.g., by an endocytosis-dependent or -independent mechanism. Ligands andassociated modifications can also increase sequence specificity andconsequently decrease off-site targeting. A tethered ligand can includeone or more modified bases or sugars that can function as intercalators.These are preferably located in an internal region, such as in a bulgeof RNA silencing agent/target duplex. The intercalator can be anaromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound.A polycyclic intercalator can have stacking capabilities, and caninclude systems with 2, 3, or 4 fused rings. The universal basesdescribed herein can be included on a ligand. In one embodiment, theligand can include a cleaving group that contributes to target geneinhibition by cleavage of the target nucleic acid. The cleaving groupcan be, for example, a bleomycin (e.g., bleomycin-A5, bleomycin-A2, orbleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), apolyamine, a tripeptide (e.g., lys-tyr-lys tripeptide), or metal ionchelating group. The metal ion chelating group can include, e.g., anLu(III) or EU(III) macrocyclic complex, a Zn(II)2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, oracridine, which can promote the selective cleavage of target RNA at thesite of the bulge by free metal ions, such as Lu(III). In someembodiments, a peptide ligand can be tethered to a RNA silencing agentto promote cleavage of the target RNA, e.g., at the bulge region. Forexample, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) canbe conjugated to a peptide (e.g., by an amino acid derivative) topromote target RNA cleavage. A tethered ligand can be an aminoglycosideligand, which can cause an RNA silencing agent to have improvedhybridization properties or improved sequence specificity. Exemplaryaminoglycosides include glycosylated polylysine, galactosylatedpolylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugatesof aminoglycosides, such as Neo-N-acridine, Neo-S-acridine,Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of anacridine analog can increase sequence specificity. For example, neomycinB has a high affinity for RNA as compared to DNA, but lowsequence-specificity. An acridine analog, neo-5-acridine has anincreased affinity for the HIV Rev-response element (RRE). In someembodiments the guanidine analog (the guanidinoglycoside) of anaminoglycoside ligand is tethered to an RNA silencing agent. In aguanidinoglycoside, the amine group on the amino acid is exchanged for aguanidine group. Attachment of a guanidine analog can enhance cellpermeability of an RNA silencing agent. A tethered ligand can be apoly-arginine peptide, peptoid or peptidomimetic, which can enhance thecellular uptake of an oligonucleotide agent.

Exemplary ligands are coupled, preferably covalently, either directly orindirectly via an intervening tether, to a ligand-conjugated carrier. Inexemplary embodiments, the ligand is attached to the carrier via anintervening tether. In exemplary embodiments, a ligand alters thedistribution, targeting or lifetime of an RNA silencing agent into whichit is incorporated. In exemplary embodiments, a ligand provides anenhanced affinity for a selected target, e.g., molecule, cell or celltype, compartment, e.g., a cellular or organ compartment, tissue, organor region of the body, as, e.g., compared to a species absent such aligand.

Exemplary ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified RNA silencing agent, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides. Ligands in general can include therapeuticmodifiers, e.g., for enhancing uptake; diagnostic compounds or reportergroups e.g., for monitoring distribution; cross-linking agents;nuclease-resistance conferring moieties; and natural or unusualnucleobases. General examples include lipophiles, lipids, steroids(e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal),carbohydrates, proteins, protein binding agents, integrin targetingmolecules, polycationics, peptides, polyamines, and peptide mimics.Ligands can include a naturally occurring substance, (e.g., human serumalbumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin orhyaluronic acid); amino acid, or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid. Examples of polyamino acids include polyaminoacid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine, multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic. Other examples of ligands include dyes,intercalating agents (e.g. acridines and substituted acridines),cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr-lystripeptide, aminoglycosides, guanidium aminoglycodies, artificialendonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol (andthio analogs thereof), cholic acid, cholanic acid, lithocholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters,e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C_(16,) C₁₇, C₁₈, C₁₉, or C₂₀ fattyacids) and ethers thereof, e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆,C_(17,) C₁₈, C₁₉, or C₂₀ alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol,1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group,hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group,palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, naproxen, vitamin E,folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-KB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the RNA silencing agent into the cell, for example, bydisrupting the cell's cytoskeleton, e.g., by disrupting the cell'smicrotubules, microfilaments, and/or intermediate filaments. The drugcan be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin. The ligand can increase the uptake of the RNAsilencing agent into the cell by activating an inflammatory response,for example. Exemplary ligands that would have such an effect includetumor necrosis factor alpha (TNFα), interleukin-1 beta, or gammainterferon. In one aspect, the ligand is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA. A lipid based ligand canbe used to modulate, e.g., control the binding of the conjugate to atarget tissue. For example, a lipid or lipid-based ligand that binds toHSA more strongly will be less likely to be targeted to the kidney andtherefore less likely to be cleared from the body. A lipid orlipid-based ligand that binds to HSA less strongly can be used to targetthe conjugate to the kidney. In a preferred embodiment, the lipid basedligand binds HSA. A lipid-based ligand can bind HSA with a sufficientaffinity such that the conjugate will be preferably distributed to anon-kidney tissue. However, it is preferred that the affinity not be sostrong that the HSA-ligand binding cannot be reversed. In anotherpreferred embodiment, the lipid based ligand binds HSA weakly or not atall, such that the conjugate will be preferably distributed to thekidney. Other moieties that target to kidney cells can also be used inplace of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics tooligonucleotide agents can affect pharmacokinetic distribution of theRNA silencing agent, such as by enhancing cellular recognition andabsorption. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long. A peptide or peptidomimetic can be, for example, a cellpermeation peptide, cationic peptide, amphipathic peptide, orhydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). Thepeptide moiety can be a dendrimer peptide, constrained peptide orcrosslinked peptide. The peptide moiety can be an L-peptide orD-peptide. In another alternative, the peptide moiety can include ahydrophobic membrane translocation sequence (MTS). A peptide orpeptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature354:82-84, 1991). In exemplary embodiments, the peptide orpeptidomimetic tethered to an RNA silencing agent via an incorporatedmonomer unit is a cell targeting peptide such as anarginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptidemoiety can range in length from about 5 amino acids to about 40 aminoacids. The peptide moieties can have a structural modification, such asto increase stability or direct conformational properties. Any of thestructural modifications described below can be utilized

EXAMPLES Example 1 Chemical Synthesis of Di-siRNAs and Vitamin DConjugated hsiRNAs

The Di-siRNAs used in the in vitro and in vivo efficacy evaluation weresynthesized as follows. As shown in FIG. 2, triethylene glycol wasreacted with acrylonitrile to introduce protected amine functionality. Abranch point was then added as a tosylated solketal, followed byreduction of the nitrile to yield a primary amine which was thenattached to vitamin D (calciferol) through a carbamate linker. The ketalwas then hydrolyzed to release the cis-diol which was selectivelyprotected at the primary hydroxyl with dimethoxytrityl (DMTr) protectinggroup, followed by succinylation with succinic anhydride. The resultingmoiety was attached to a solid support followed by solid phaseoligonucleotide synthesis and deprotection resulting in the threeproducts shown; VitD, Capped linker, and Di-siRNA. The products ofsynthesis were then analyzed as described in Example 4.

Example 2 Alternative Synthesis Route 1

As shown in FIG. 5, the mono-phosphoamidate linker approach involves thefollowing steps: Mono-azide tetraethylene glycol has a branch pointadded as a tosylated solketal. The ketal is then removed to release thecis-diol which is selectively protected at the primary hydroxyl withdimethoxytrityl (DMTr) protecting group, followed by reduction of theazide by triphenylphosphine to a primary amine, which is immediatelyprotected with a monomethoxy trityl (MMTr) protecting group. Theremaining hydroxyl is succinylated with succinic anhydride and coupledto solid support (LCAA CPG). Oligonucleotide synthesis and deprotectionaffords one main product the, the di-siRNA with a phosphate andphosphoamidate linkage. This example highlights an alternative anddirect route of synthesis to produce solely the phosphate andphosphoamidate linker.

Example 3 Alternative Synthesis Route 2

In order to produce a di-phosphate containing moiety, a secondalternative synthesis approach was developed. As shown in FIG. 5, thedi-phosphoate linker approach involves the following steps: Startingfrom a solketal-modified teraethylene glycol, the ketal is removed andthe two primary hydroxyls are selectively protected with dimethoxytrityl (DMTr). The remaining hydroxyl is extended in length with a silylprotected 1-bromoethanol. The TBDMS is removed, succinylated andattached to solid support. This is followed by solid phaseoligonucleotide synthesis and deprotection, producing the Di-siRNA withthe diphosphate containing linker.

Example 4 Quality Control of Chemical Synthesis of Di-siRNAs and VitaminD Conjugated hsiRNAs. HPLC

To assess the quality of the chemical synthesis of Di-siRNAs and VitaminD conjugated hsiRNAs, analytical HPLC was used to identify and quantifythe synthesized products. Three major products were identified: thesiRNA sense strand capped with a tryethylene glycol (TEG) linker, theDi-siRNA, and the vitamin D conjugated siRNA sense strand (FIG. 3). Eachproduct was isolated by HPLC and used for subsequent experiments. Thechemical structures of the three major products of synthesis are shownin FIG. 3. The conditions for HPLC included: 5-80% B over 15 minutes,Buffer A (0.1 M TEAA+5% ACN), Buffer B (100% ACN).

Mass Spectrometry

Further quality control was done by mass spectrometry, which confirmedthe identity of the Di-siRNA complex. The product was observed to have amass of 11683 m/z, which corresponds to two sense strands of the siRNAattached at the 3′ ends through the TEG linker (FIG. 4). In thisspecific example the siRNA sense strand was designed to target theHuntingtin gene (Htt). The method of chemical synthesis outlined inExample 1 successfully produced the desired product of a Di-branchedsiRNA complex targeting the Huntingtin gene. LC-MS conditions included:0-100% B over 7 minutes, 0.6 mL/min. Buffer A (25 mM HFIP, 15 mM DBA in20% MeOH), Buffer B (MeOH with 20% Buffer A).

Example 5 Efficacy and Cellular Uptake of By-products of ChemicalSynthesis of Di-siRNAs and Vitamin D Conjugated hsiRNAs

To assess the Htt gene silencing efficacy of each HPLC-isolatedby-product of the chemical synthesis of Di-siRNAs and Vitamin Dconjugated hsiRNAs, HeLa cells were treated with each isolated productby lipid-mediated transfection. Huntingtin mRNA expression was assessedthrough Affymetrix Quantigene 2.0 and normalized to a housekeeping gene(PPIB). All four by-products resulted in significant Htt gene silencing72 hours post transfection (FIG. 22). Cellular uptake was tested in vivoby delivering each fluorescently labeled by-product to mice viainstrastriatal injection and measuring the uptake by fluorescentimaging. The Di-siRNA product showed dramatically increased uptake inthe injected hemisphere of the mouse brain compared to the other threeby-products (FIG. 22). Of the four by-products resulting from thechemical synthesis reaction, the Di-siRNA shows both efficientgene-silencing and high levels of cellular uptake in vivo.

Example 6 In Vitro Efficacy of Di-branched siRNA Structure

To determine the in vitro efficacy of Di-branched siRNAs (Di-siRNAs),Di-siRNAs targeting Htt were transfected into HeLa cells using alipid-mediated delivery system. HeLa cells were transfected withbranched oligonucleotides at varying concentrations using RNAiMax. HTTmRNA expression was measured 72 hours after transfection. The Di-siRNAscaused significant silencing of the HTT gene, similar to the effectresulting from single siRNA duplex in HeLa cells (FIG. 10).

To determine efficiency of cellular uptake and gene silencing in primarycortical neurons without lipid mediated delivery, cells were treatedpassively with Htt-Di-siRNAs at varying concentrations for one week. HTTmRNA expression was measured and normalized to the housekeeping genePPIB. As shown in FIG. 10, the Di-siRNA structure led to significantsilencing of the Htt gene, showing that the Di-branched siRNA structureis efficiently delivered to neurons without the lipid formulation. Thisdemonstrates that the Di-branched structure of the siRNA complex doesnot hinder RISC loading and the gene silencing effects of knowneffective siRNAs.

Example 7 Route of Administration of Di-siRNAs and Vitamin D ConjugatedhsiRNAs

To assess the efficacy of delivery and activity of branchedoligonucleotides in vivo in neurons, Di-HTT-Cy3 was delivered to micevia intrastriatal (IS) injection. Di-HTT-Cy3 localized to andaccumulated throughout the injected hemisphere of the brain, whereas thesingle branch HTT-siRNA (tryethylene glycol conjugated siRNA(TEG-siRNA)) showed significantly lower accumulation in the injectedhemisphere of the brain (FIG. 11). A single IS injection of Htt-Di-siRNAresulted in significant gene silencing one week post injection (FIG. 13)and the level of gene silencing was maintained two weeks post injection(FIG. 26). Further experiments showed that a single IS injection ofDi-HTT-Cy3 did not result in significant toxicity two weeks postinjection (FIG. 27A). However, the Htt-Di-siRNAs did cause significantgliosis (FIG. 27B), which is to be expected when the Htt gene issilenced in neurons. Furthermore, the Di-HTT-Cy3 does not accumulate inthe liver or kidney two weeks post IS injection (FIG. 13), nor is theHtt mRNA significantly silenced in the liver or kidney following ISinjection (FIG. 23). The double-branch structure of the Di-siRNAssignificantly improves distribution and neuronal uptake when comparedwith the TEG-siRNA only; therefore it is likely that the size and/or thestructure of the siRNA complex are important for efficacy. The ISinjection of Htt-Di-siRNAs leads to significant and stable depletion ofHtt, which stays localized to the brain, this level of efficacy hasnever been demonstrated for non-conjugated siRNAs.

Example 8 Alternative Route of Administration 1

To assess the efficacy of delivery and activity of branchedoligonucleotides in the spinal cord, Di-HTT-Cy3 was delivered to micevia intrathecal (IT) injection in the lumbar region of the spinal cord.As shown in FIGS. 14 and 28-29, Di-HTT-Cy3 accumulated in the spinalcord one week post injection. IT injection also led to significant HttmRNA silencing in the cervical, thoracic, and lumbar regions of thespinal cord one week post injection (FIG. 14). The IT injection ofDi-HTT-Cy3 successfully led to significant gene silencing in the spinalcord.

Example 9 Alternative Route of Administration 2

To assess the efficacy of delivery and activity of branchedoligonucleotides throughout the brain in a clinically relevantexperiment, Di-HTT-Cy3 was delivered to mice via intracerebroventricular(ICV) injection. The Di-siRNAs accumulated throughout the brain at bothtwo days and two weeks post injection (FIGS. 30-31). ICV injection ofDi-HTT-Cy3 also significantly silenced Htt mRNA and protein expressiontwo weeks post injection (FIG. 32). Further experiments showed the ICVdelivery did not result in significant toxicity two weeks post injection(FIG. 33). However, ICV injection of Di-HTT-Cy3 did result insignificant gliosis in multiple areas of the brain, which is an expectedresult upon silencing of the Htt gene (FIG. 34). The ICV injectiondirectly administers the Di-siRNAs to the cerebrospinal fluid (CSF) inorder to bypass the blood brain barrier and this injection is used totreat diseases of the brain.

This result is important for the therapeutic potential of branchedoligonucleotides, as ICV injection is a therapeutically relevantinjection for neurological diseases. The efficacy and stability of thebranched oligonucleotides following ICV administration demonstrates thatthe invention described herein could be utilized as therapy in a varietyof hard to treat neurological diseases, including Huntington's disease.

Example 10 Alternative Route of Administration 3

To assess the efficacy of delivery and activity of Di-siRNAs throughoutthe body, Di-HTT-Cy3 was administered to mice via intravenous (IV)injection. The mice were injected with 20 mg/kg Di-HTT-Cy3 and twoconsecutive days (total of 40 mg/kg) and were sacrificed 24 hours afterthe final injection. As shown in FIGS. 35-36, the Di-siRNAs accumulatedin multiple organs (including liver, kidney, spleen, pancreas, lung,fat, muscle, thymus, colon, and skin) following IV delivery. TheDi-siRNAs also accumulated in the brain, demonstrating the ability ofthe Di-siRNAs to cross the blood-brain barrier, an unprecedented resultusing therapeutic siRNAs. The IV injection demonstrates that theDi-siRNA structure is effective and functional in a wide variety of celltypes throughout the body.

Example 11 Determination of Toxicity and Gliosis Toxicity

In order to assess the level of toxicity in the brain followinginjection of Di-HTT-Cy3, protein levels of DARPP32 were assessed inbrain tissue because elevated DARPP32 indicates neuronal death (Jin, H.,et al. DARPP-32 to quantify intracerebral hemorrhage-induced neuronaldeath in basal ganglia. Transl Stroke Res. 4(1): 130-134. 2013). Micewere treated with 2 nmols Di-HTT-Cy3 (4 nmols of corresponding antisenseHTT strand) via IS or ICV injection. The animals were sacrificed 14 daysafter injection and tissue punches were taken from 300 μm brain slicesfrom different areas of the brain. DARPP32 protein was quantified byimmunoblot. Artificial cerebrospinal fluid (aCSF) was used as a negativecontrol. Neither IS nor ICV injection of high dose Di-HTT-Cy3 resultedin significant toxicity (FIGS. 27 and 33).

Gliosis

In order to assess the level of gliosis in the brain following injectionof Di-HTT-Cy3, GFAP protein levels were assessed following high dose ofDi-HTT-Cy3. Mice were treated with 2 nmols Di-HTT-Cy3 (4 nmols ofcorresponding antisense HTT strand) via IS or ICV injection. The animalswere sacrificed 14 days after injection and tissue punches were takenfrom 300 μm brain slices from different areas of the brain. GFAP proteinwas quantified by immunoblot. Artificial cerebrospinal fluid (aCSF) wasused as a negative control. Artificial cerebrospinal fluid (aCSF) wasused as a negative control. Both IS and ICV injection of high doseDi-HTT-Cy3 resulted in significant gliosis (FIGS. 27 and 34), howeverinduction of gliosis is an expected result upon near complete silencingof the Huntingtin gene.

Example 12 Determination of Di-HTT-Cy3 Efficacy In Vivo Distribution andAccumulation

In order to determine the efficacy of distribution of branchedoligonucleotides in vivo, mice were treated with Di-HTT-Cy3 via IS, ICV,intrathecal, or IV injections as described above in Examples 7-10. Inall Examples, 2 nmols Di-HTT-Cy3 (4 nmols of corresponding antisense HTTstrand) was injected and accumulation was quantified by usingCy3-labeled peptide nucleic acids (PNAs) to hybridize to the sensestrand. HPLC analysis was then used to quantify ng of Di-HTT-Cy3 per mgof tissue. Artificial cerebrospinal fluid (aCSF) was used as a negativecontrol.

In fluorescent imaging experiments, brain slices were stained with DAPI(blue) imaged using the Cy3 channel to detect accumulation of Di-HTT-Cy3(red).

Silencing

In order to determine the efficacy of silencing of branchedoligonucleotides in vivo, mice were treated with Di-HTT-Cy3 via IS, ICV,intrathecal, or IV injections as described above in Examples 7-10. Inall examples, 2 nmols Di-HTT-Cy3 (4 nmols of corresponding antisense HTTstrand) was injected and silencing of Htt mRNA was quantified usingAffymetrix Quantigene 2.0 as described in Coles, A. et al., AHigh-Throughput Method for Direct Detection of TherapeuticOligonucleotide-Induced Gene Silencing In Vivo. Nucl Acid Ther. 26 (2),86-92, 2015. Data was normalized to the housekeeping control, HPRT andartificial cerebrospinal fluid (aCSF) was used as a negative control.

Example 13 Incorporation of a Hydrophobic Moiety in the BranchedOligonucleotide Structure: Strategy 1

In one example, a short hydrophobic alkylene or alkane (Hy) with anunprotected hydroxyl group (or amine) that can be phosphitylated with2-Cyanoethoxy-bis(N,N-diisopropylamino)phosphine (or any other suitablephosphitylating reagent) is used to produce the corresponding lipophilicphosphoramidite. These lipophilic phosphoramidites can be added to theterminal position of the branched oligonucleotide using conventionaloligonucleotide synthesis conditions. This strategy is depicted in FIG.44.

Example 14 Incorporation of a Hydrophobic Moeity in the BranchedOligonucleotide Structure: Strategy 2

In another example, a short/small aromatic planar molecule (Hy) that hasan unprotected hydroxyl group with or without a positive charge (oramine) that can be phosphitylated with2-Cyanoethoxy-bis(N,N-diisopropylamino)phosphine (or any other suitablephosphitylating reagent) is used to produce the corresponding aromatichydrophobic phosphoramidite. The aromatic moiety can have a positivecharge. These lipophilic phosphoramidites can be added to the terminalposition of the branched oligonucleotide using conventionaloligonucleotide synthesis conditions. This strategy is depicted in FIG.45.

Example 15 Incorporation of a Hydrophobic Moeity in the BranchedOligonucleotide Structure: Strategy 3

To introduce biologically relevant hydrophobic moieties, shortlipophilic peptides are made by sequential peptide synthesis either onsolid support or in solution (the latter being described here). Theshort (1-10) amino acid chain can contain positively charged or polaramino acid moieties as well, as any positive charge will reduce theoverall net charge of the oligonucleotide, therefore increasing thehydrophobicity. Once the peptide of appropriate length is made it shouldbe capped with acetic anhydride or another short aliphatic acid toincrease hydrophobicity and mask the free amine. The carbonyl protectinggroup is then removed to allow for 3-aminopropan-1-ol to be coupledallowing a free hydroxyl (or amine) to be phosphitylated. This aminoacid phosphoramidite can then be added to the terminal 5′ position ofthe branched oligonucleotide using conventional oligonucleotidesynthesis conditions. This strategy is depicted in FIG. 46.

1-30. (canceled)
 31. A method of selectively delivering a nucleic acidto a target organ, the nucleic acid having the structure of formula(VIII):L—(cNA)_(n)  (VIII) wherein L is selected from an ethylene glycol chain,an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, aphosphoramidate, an ester, an amide, a triazole, and combinationsthereof, wherein formula (VIII) optionally further comprises one or morebranch point B, and one or more spacer S, wherein B is independently foreach occurrence a polyvalent organic species or derivative thereof; S isindependently for each occurrence selected from an ethylene glycolchain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate,a phosphoramidate, an ester, an amide, a triazole, and combinationsthereof; each cNA, independently, is a carrier nucleic acid comprisingone or more chemical modifications; and n is 2, 3, 4, 5, 6, 7 or
 8. 32.The method of claim 31, having a structure selected from formulas(VIII-1)-(VIII-9):


33. The method of claim 31, wherein each cNA independently comprises atleast 15 contiguous nucleotides.
 34. The method of claim 31, whereineach cNA independently consists of chemically-modified nucleotides. 35.The method of claim 31, further comprising n therapeutic nucleic acids(NA), wherein each NA is hybridized to at least one cNA.
 36. The methodof claim 35, wherein each NA independently comprises at least 16contiguous nucleotides.
 37. The method of claim 35, wherein each NAindependently comprises 16-20 contiguous nucleotides.
 38. The method ofclaim 35, wherein each NA comprises an unpaired overhang of at least 2nucleotides.
 39. The method of claim 35, wherein the nucleotides of theoverhang are connected via phosphorothioate linkages.
 40. The method ofclaim 35, wherein each NA, independently, is selected from the groupconsisting of: DNA, siRNAs, antagomiRs, miRNAs, gapmers, mixmers, orguide RNAs.
 41. The method of claim 35, wherein each NA is the same. 42.The method of claim 35, wherein each NA is not the same.
 43. (canceled)44. The method of claim 31, wherein the target of delivery is selectedfrom the group consisting of: brain, spinal cord, liver, skin, kidney,spleen, pancreas, colon, fat, lung, muscle, and thymus.
 45. The methodof claim 31, wherein the target of delivery is the brain.
 46. The methodof claim 45, wherein the target of delivery is one or both of thestriatum of the brain and the cortex of the brain.
 47. The method ofclaim 31, further comprising a hydrophobic moiety attached to theterminal 5′position of the nucleic acid.
 48. The method of claim 47,wherein the hydrophobic moiety comprises an alkyl, alkenyl, or arylmoiety, a vitamin or cholesterol derivative, a lipophilic amino acid, ora combination thereof.